Methods and means for inhibition of CDK4 activity

ABSTRACT

p 21   WAF1  interacts with cyclin D1 and Cdk4. Peptide fragments of p21 inhibit the interaction and/or affect Cdk4 activity. The peptides, derivative peptides and non-peptidyl mimetics thereof are useful in affecting activity of Cdk4, such as RB phosphorylation, and cellular proliferation, indicative of therapeutic usefulness in treatment of tumours and other hyperproliferative disorders. Assay and screening methods allow identification of such modulators, especially inhibitors, of Cdk4 activity.

[0001] The present invention relates to substances and their therapeuticuse, and in particular to the identification of regions of p₂₁ ^(WAF1)that bind to cyclin dependent kinases, specifically Cdk4, and/or cyclinD1, and to substances, fragments and mimetics based on this region. Thepresent invention also relates to pharmaceutical compositions comprisingthese molecules and their use in therapeutic applications for treatinghyperproliferative disorders, such as cancer and psoriasis, andcompositions comprising these Molecules and their use in applicationsrelating to growth in eukaryotic cells. The invention also relates toassay methods and means for identifying substances useful forinterfering with p21/Cdk4/cyclin interaction, and preferably inhibitingCdk4 activity.

[0002] The tumour suppressor function of p53 is linked to a DNA-damageinducible cell cycle checkpoint pathway (Kastan et al., 1991), in whichp53 can induce either growth arrest (Agarwal et al., 1995) or apoptosis(Clarke et al., 1993; Lowe et al., 1993; Merritt et al., 1994) in thedamaged cells. The biochemical activity of p53 most tightly associatedwith tumour suppression and growth arrest involves an ionisingradiation-dependent activation of sequence-specific transcriptionalactivity (Kastan et al., 1991; Lu and Lane, 1993; Pietenpol, et al.,1994). p53 induces the transcription of a number of genes, the productsof which play a direct role in mediating growth arrest. Thesep53-inducible negative regulators of cell proliferation include: thecyclin dependent kinase inhibitor (CKI), p21^(WAF1) (El-Deiry et al.,1993; Harper et al., 1993; Xiong et al., 1993; Gu et al., 1993); ranapoptosis promoting protein, Bax (Miyashita and Reed, 1995); the insulingrowth factor binding protein IGF-BP3 (Buckbinder et al., 1995); andGadd45 (Kastan et al., 1992), a potent inhibitor of cell proliferationwith an as yet unclear biochemical function (Kearsey et al., 1995).

[0003] A common event in the development of human neoplasia is theinactivation of a DNA damage-inducible cell cycle checkpoint pathwayregulated by p53 (Hollstein et al., 1991; Lane, 1992; Agrawal et al.,1995) A variety of mechanisms can lead to the functional inactivation ofthe p53 pathway, including: missense mutations within, or deletions ofthe p53 gene, inactivation of wild type p53 protein function byinteraction with the oncogenic cellular protein mdm-2 (Momand et al.,1992), or the inability to induce downstream effector molecules, such asp21^(WAF1) (Deng et al., 1995; Waldman et al., 1995).

[0004] Our growing knowledge of the molecular mechanisms underlying thetransformation of mammalian cells offers the opportunity to createrationally designed inhibitors of specific biochemical processesessential to uncontrolled cell proliferation or cancer. Recentdevelopments have shown that the reactivation of the p53 pathway in somehuman tumours could in theory be achieved by: (i) activating thebiochemical function of mutant p53 protein (Halazonetis and Kandil,1993; Hupp et al., 1993), possibly using small peptides as leads fordrug design (Hupp et al., 1995); (ii) disrupting the interaction of theoncogene mdm-2 and wild type p53 through the use of peptide-mimeticinhibitors of complex formation (Picksley et al., 1994); (iii) restoringor mimicking the function of the downstream effector moleculep21^(WAF1), which on its own is capable of mediating growth suppression(El-Deiry et al., 1993; Eastham et al., 1995).

[0005] p21^(WAF1) is an inhibitor of both the G1 cyclin dependentprotein kinases (CDKS; which control the progression from G1 into Sphase) (Harper et al., 1995) and proliferating cell nuclear antigen(PCNA; an essential DNA-replication factor) (Florez-Rozas et al., 1994;Waga et al., 1994). Thus, inhibition of the function of either CDKs orPCNA provides, in theory, two distinct avenues for development of drugdiscovery programmes which are based on the activity of p21^(WAF1). ThePCNA binding function of p21^(WAF1) can be mimicked by a 20-amino acidpeptide derived from the C-terminal domain of p21^(WAF1) and thispeptide is sufficient to partially inhibit SV40 replication in vitro(Warbrick et al., 1995).

[0006] Despite its PCNA binding role, the primary function of thep21^(WAF1) protein as a growth suppressor appears to be inhibition ofthe G1 cyclin-CDK complexes (Chen et al., 1995; Harper et al., 1995; Luoet al., 1995; Nakanishi et al., 1995b). Luo et al. (1995) reported theN-terminal domain of p21, composed of residues 1-75, to act as aCDK-inhibitor in vitro, inhibiting cyclin E-Cdk2.

[0007] The present invention concerns (i) the elucidation of themolecular mechanism of cyclin D1 -Cdk4 complex inhibition by p21^(WAF1),and (ii) the identification of peptide mimetics of p21^(WAF1) inhibitoryactivity, through the examination of the binding and inhibitoryproperties of a series of synthetic peptides based on the amino acidsequence of p21^(WAF1). Our studies found that two peptides derived fromthe N-terminal domain of p21^(WAF1) have biochemical activity; a peptide4 (residues 46-65) forms a stable complex with Cdk4, but has noinhibitory activity, while a peptide 2 (residues 16-35) binds to cyclinD1 and inhibits Cdk4 activity with a I_(0.5) of 2 μM.

[0008] These data define for a cyclin binding site on p21^(WAF1) andsuggest that one mechanism involved in the CDK inhibitory action ofp21^(WAF1) employs binding to the cyclin subunit of the CDK holoenzyme.This has lead us to propose that p21^(WAF1) can inhibit Cdk4 activityallosterically through conformational or (ii) interfering with thecyclin-Cdk interaction or (iii) interfering with thecyclin-Cdk-substrate interaction changes in the structure of cyclin D1.Furthermore, peptides based on the C-terminal sequence of p21^(WAF1)interact with both cyclin D1 and Cdk4, and are potent inhibitors of Cdk4activity, with a peptide (peptide 10 herein) composed of residues141-160 having an I_(0.5) of 0.1 μM. We show that both of the inhibitorypeptides bind at physiologically relevant sites on cyclin D1 and/orCdk4, and that they display specificity mimicking that of full lengthp21^(WAF1). Importantly, the potency of the C-terminal peptide isimproved by making a single amino acid substitution (D-A at position149). We have mapped the inhibitory component of this peptide usingalanine mutation analysis and show that it is distinct from the PCNAinteraction domain, which also resides in the C-terminal region of thep21^(WAF1) protein.

[0009] Remarkably, a stretch of just five amino acids contains the Cdk4inhibitory motif and a single conservative mutation at either of twohydrophobic amino acid residues completely abolishes the inhibitoryactivity of the peptide. These data have exciting implications for themechanism of action of p21^(WAF1) protein and represent a starting pointfor a drug design programme aimed at producing synthetic moleculesfunctioning as tumour suppressors downstream of p53.

[0010] Accordingly, in one aspect, the present invention provides asubstance which has the property of inhibiting Cdk4, said substancecomprising:

[0011] (i) a peptide fragment consisting of the motif xyLzF, wherein yand z are any amino acid and x is preferably R, or a derivative of saidpeptide fragment; or,

[0012] (ii) a functional mimetic of said peptide fragment.

[0013] In a further aspect, the present invention provides the abovesubstance for use in a method of medical treatment.

[0014] In a further aspect, the present invention provides the use of asubstance which has the property of inhibiting Cdk4 in the preparationof a medicament for the treatment of a hyperproliferative disorder, saidsubstance comprising:

[0015] (i) fragment of the C-terminal portion of p21^(WAF1), or anactive portion or derivative thereof; or,

[0016] (ii) a peptide fragment including the motif xyLzF, wherein y andz are any amino acid and x is preferably R, or a derivative of saidpeptide fragment; or,

[0017] (iii) a functional mimetic of (i) or (ii).

[0018] In a preferred embodiment, the C-terminal portion of p21^(WAF1)consisting of the peptide motif KRRLIFSK was found to completely inhibitcyclin-Cdk4 activity and to prevent phosphorylation of pRb (see FIG. 6).

[0019] In a further aspect, the present invention provides a substancewhich has the property of binding to Cdk4 for use in a method of medicaltreatment, said substance comprising:

[0020] (i) a fragment of the p21^(WAF1) protein consisting of residues46-65 of the p21^(WAF1) amino acid sequence, or an active portion orderivative thereof; or,

[0021] (ii) a functional mimetic of said fragment.

[0022] In a further aspect, the present invention provides the use of asubstance which has the property of binding Cdk4 in the preparation of amedicament for the treatment of a hyperproliferative disorder, saidsubstance comprising:

[0023] (i) a fragment of the p21^(WAF1) protein consisting of residues46-65 of the p21^(WAF1) amino acid sequence, or an active portion orderivative thereof; or,

[0024] (ii) a functional mimetic of said fragment.

[0025] In a further aspect, the present invention provides a substancewhich has the properties of binding cyclin D and/or inhibiting Cdk4 foruse in a method of medical treatment, said substance comprising:

[0026] (i) a fragment of the p21^(WAF1) protein consisting of residues16-35 of the p21^(WAF1) amino acid sequence, or an active portion orderivative thereof; or,

[0027] (ii) a functional mimetic of said fragment.

[0028] In a further aspect, the present invention provides the use of asubstance which has the property of binding cyclin D1 and/or inhibitingCdk4 in the preparation of a medicament for the treatment of ahyperproliferative disorder, said substance comprising:

[0029] (i) a fragment of the p21^(WAF1) protein consisting of residues16-35 of the p21^(WAF1) amino acid sequence, or an active portion orderivative thereof; or,

[0030] (ii) a functional mimetic of said peptide fragment.

[0031] Based on experimental evidence included below showing residuesinvolved in binding of peptide 2 (residues 16-35), and the crystalstructure available for p27 (related to p21), the following generalformula for peptides useful in accordance with various aspects of thepresent invention is provided:

KxxRRyFzP

[0032] wherein x may be any amino acid, y and z may be hydrophobic, andeach of the underlined residues may be absent or different, i.e. anotheramino acid. Hydrophobic residues may be alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan, methionine. Either orboth of the amino acids R may be substituted by other basic residues,particularly lysine (K) or histidine (H).

[0033] In the present invention, “an active portion” means a peptidewhich is less than the fragment of the p21^(WAF1) amino acid sequence,but which retains the relevant property mentioned above.

[0034] In the present invention, “functional mimetic” means a substancewhich may not contain an active portion of the p21^(WAF1) amino acidsequence and is probably not a peptide at all, but which has therelevant property mentioned above.

[0035] In the present invention, “a derivative” means a peptide modifiedby varying its amino acid sequence, eg by manipulation of the nucleicacid encoding the peptide or by altering the peptide itself. Suchderivatives of the natural amino acid sequence may involve insertion,addition, deletion or substitution of one or more amino acids, withoutfundamentally altering the essential activity of the peptides. Anexample of a derivative is the p21^(WAF1) mutant in which A wassubstituted for D at position 149 of the full length protein, thismutant having enhanced cyclin D1 -Cdk4 inhibitory activity.

[0036] Preferred substances according to certain embodiments of thepresent invention do not bind PCNA, and/or do not interfere with p21interaction or binding with PCNA.

[0037] Cell cycle arrest may be induced by various aspects according tothe present invention in Rb negative and/or Rb positive cells, asexemplified experimentally below.

[0038] In a further aspect, the present invention providespharmaceutical compositions comprising one or more of the abovesubstances in combination with a pharmaceutically acceptable carrier.

[0039] In a further aspect, the present invention relates tocompositions comprising one or more of the above substances and theiruse in controlling the growth of eukaryotic cells, eg as a foodpreservative or as an agent to promote the growth of plants.

[0040] In a further aspect, the present invention provides compoundscomprising any of the above substances coupled to carrier molecules,enabling the compounds to be delivered to cells in vivo. In oneembodiment, the carrier molecule is a 16 aa peptide sequence derivedfrom the homeodomain of Antennapedia (e.g. as sold under the name“Penetratin”) which can be coupled to one of the above substances via aterminal Cys residue. Alternatively, as in the examples described below,a carrier peptide (having the sequence RQIKIWFQNRRMKWKK) can besynthesised so it is directly attached to peptide fragments. The“Penetratin” molecule and its properties are described in WO91/18981.

[0041] Thus, the present invention in various aspects provides forinterfering with or interrupting interaction between p21 and cyclin D1and/or Cdk4 using an appropriate agent.

[0042] Such an agent may be capable of blocking binding between a sitelocated at amino acid residues identified herein as being involved inand/or important for binding or interaction with cyclin D1 and/or Cdk4.

[0043] The full sequence of the p21 protein has been elucidated and isset out in WO95/13375, WO93/12251 and WO95/06415 which are incorporatedherein by reference.

[0044] Such agents may be identified by screening techniques whichinvolve determining whether an agent under test inhibits or disrupts thebinding of p21 protein or a suitable fragment, derivative, analogue orfunctional mimetic thereof, with cyclin D1 and/or Cdk4, or a relevantfragment, derivative, analogue or functional mimetic thereof.

[0045] Suitable fragments of p21 include those which include residues asidentified herein. Smaller fragments, and derivatives, analogues andfunctional mimetics of this fragment may similarly be employed, e.g.peptides identified using a technique such as alanine scanning.

[0046] In a further aspect of the invention, whereas assays using thepeptides described herein and the use of the peptides are described inthe context of modulating the interaction of p21 with cyclin D1 and/orCdk4, these peptides may also be useful as p21 mimetics to inhibit theinteraction of p21 and other cyclin-Cdk interactions, particularly G1complexes such as cyclin E-Cdk2. Thus the various described embodimentsof the invention above and below herein with regard to cyclin D1 and/orCdk4 is applicable mutatis mutandis to these other cyclins and/or Cdksrespectively.

[0047] Screening methods and assays are discussed in further detailbelow.

[0048] One class of agents that can be used to disrupt the binding ofp21 and cyclin D1 and/or Cdk4 are peptides based on the sequence motifsof p21 that interact with cyclin D1 and/or Cdk4. Such peptides tend tobe small molecules, and may be about 40 amino acids in length or less,preferably about 35 amino acids in length or less, more preferably about30 amino acids in length, or less, more preferably about 25 amino acidsor less, more preferably about 20 amino acids or less, more preferablyabout 15 amino acids or less, more preferably about 10 amino acids orless, or 9, 8, 7, 6 5 or less in length. The present invention alsoencompasses peptides which are sequence variants or derivatives of awild type p21 sequence.

[0049] Preferably, the amino acid sequence shares homology with afragment of the relevant p21 fragment sequence shown preferably at leastabout 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%homology, or at least about 90% or 95% homology. Thus, a peptidefragment of p21 may include 1, 2, 3, 4, 5, greater than 5, or greaterthan 10 amino acid alterations such as substitutions with respect to thewild-type sequence.

[0050] A derivative of a peptide for which the specific sequence isdisclosed herein may be in certain embodiments the same length orshorter than the specific peptide. In other embodiments the peptidesequence or a variant thereof may be included in a larger peptide, asdiscussed above, which may or may not include an additional portion ofp21. 1, 2, 3, 4 or 5 or more additional amino acids, adjacent to therelevant specific peptide fragment in p21, or heterologous thereto maybe included at one end or both ends of the peptide.

[0051] As is well-understood, homology at the amino acid level isgenerally in terms of amino acid similarity or identity. Similarityallows for “conservative variation”, i.e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine. Similarity may be as defined and determined by the TBLASTNprogram, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, which isin standard use in the art. Homology may be over the full-length of therelevant peptide or over a contiguous sequence of about 5, 10, 15, 20,25, 30 or 35 amino acids, compared with the relevant wild-type aminoacid sequence.

[0052] As noted, variant peptide sequences and peptide and non-peptideanalogues and mimetics may be employed, as discussed further below.

[0053] Various aspects of the present invention provide a substance,which may be a single molecule or a composition including two or morecomponents, which includes a peptide fragment of p21 which includes asequence as recited above and/or disclosed elsewhere herein, a peptideconsisting essentially of such a sequence, a peptide including avariant, derivative or analogue sequence, or a non-peptide analogue ormimetic which has the ability to bind cyclin D1 and/or Cdk4 and/ordisrupt or interfere with interaction between p21 and cyclin D1 and/orCdk 4.

[0054] Variants include peptides in which individual amino acids can besubstituted by other amino acids which are closely related as isunderstood in the art and indicated above.

[0055] Non-Peptide Mimetics of Peptides are Discussed Further Below.

[0056] As noted, a peptide according to the present invention and foruse in various aspects of the present invention may include or consistessentially of a fragment of p21 as disclosed, such as a fragment whosesequence is given above. Where one or more additional amino acids areincluded, such amino acids may be from p21 or may be heterologous orforeign to p21. A peptide may also be included within a larger fusionprotein, particularly where the peptide is fused to a non-p21 (i.e.heterologous or foreign) sequence, such as a polypeptide or proteindomain.

[0057] The invention also includes derivatives of the peptides,including the peptide linked to a coupling partner, e.g. an effectormolecule, a label, a drug, a toxin and/or a carrier or transportmolecule. Techniques for coupling the peptides of the invention to bothpeptidyl and non-peptidyl coupling partners are well known in the art.In one embodiment, the carrier molecule is a 16 aa peptide sequencederived from the homeodomain of Antennapedia (e.g. as sold under thename “Penetratin”), which can be coupled to a peptide via a terminal Cysresidue. The “Penetratin” molecule and its properties are described inWO 91/18981.

[0058] Peptides may be generated wholly or partly by chemical synthesis.The compounds of the present invention can be readily prepared accordingto well-established, standard liquid or, preferably, solid-phase peptidesynthesis methods, general descriptions of which are broadly available(see, for example, in J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984),in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis,Springer Verlag, N.Y.(1984); and Applied Biosystems 430A Users Manual,ABI Inc., Foster City, Calif.), or they may be prepared in solution, bythe liquid phase method or by any combination of solid-phase, liquidphase and solution chemistry, e.g. by first completing the respectivepeptide portion and then, if desired and appropriate, after removal ofany protecting groups being present, by introduction of the residue X byreaction of the respective carbonic or sulfonic acid or a reactivederivative thereof.

[0059] Another convenient way of producing a peptidyl molecule accordingto the present invention (peptide or polypeptide) is to express nucleicacid encoding it, by use of nucleic acid in an expression system.

[0060] Accordingly the present invention also provides in variousaspects nucleic acid encoding the polypeptides and peptides of theinvention.

[0061] Generally, nucleic acid according to the present invention isprovided as an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression. Nucleic acid may be wholly or partially synthetic and mayinclude genomic DNA, cDNA or RNA. Where nucleic acid according to theinvention includes RNA, reference to the sequence shown should beconstrued as reference to the RNA equivalent, with U substituted for T.

[0062] Nucleic acid sequences encoding a polypeptide or peptide inaccordance with the present invention can be readily prepared by theskilled person using the information and references contained herein andtechniques known in the art (for example, see Sambrook, Fritsch andManiatis, “Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989, and Ausubel et al, Short Protocols in MolecularBiology, John Wiley and Sons, 1992), given the nucleic acid sequence andclones available. These techniques include (i) the use of the polymerasechain reaction (PCR) to amplify samples of such nucleic acid, e.g. fromgenomic sources, (ii) chemical synthesis, or (iii) preparing cDNAsequences. DNA encoding p21 fragments may be generated and used in anysuitable way known to those of skill in the art, including by takingencoding DNA, identifying suitable restriction enzyme recognition siteseither side of the portion to be expressed, and cutting out said portionfrom the DNA. The portion may then be operably linked to a suitablepromoter in a standard commercially available expression system. Anotherrecombinant approach is to amplify the relevant portion of the DNA withsuitable PCR primers.

[0063] Modifications to the p21 sequences can be made, e.g. using Sitedirected mutagenesis, to lead to the expression of modified p21 peptideor to take account of codon preference in the host cells used to expressthe nucleic acid.

[0064] In order to obtain expression of the nucleic acid sequences, thesequences can be incorporated in a vector having one or more controlsequences operably linked to the nucleic acid to control its expression.The vectors may include other sequences such as promoters or enhancersto drive the expression of the inserted nucleic acid, nucleic acidsequences so that the polypeptide or peptide is produced as a fusionand/or nucleic acid encoding secretion signals so that the polypeptideproduced in the host cell is secreted from the cell. Polypeptide canthen be obtained by transforming the vectors into host cells in whichthe vector is functional, culturing the host cells so that thepolypeptide is produced and recovering the polypeptide from the hostcells or the surrounding medium. Prokaryotic and eukaryotic cells areused for this purpose in the art, including strains of E. coli, yeast,and eukaryotic cells such as COS or CHO cells.

[0065] Thus, the present invention also encompasses a method of making apolypeptide or peptide (as disclosed), the method including expressionfrom nucleic acid encoding the polypeptide or peptide (generally nucleicacid according to the invention). This may conveniently be achieved bygrowing a host cell in culture, containing such a vector, underappropriate conditions which cause or allow expression of thepolypeptide. Polypeptides and peptides may also be expressed in in vitrosystems, such as reticulocyte lysate.

[0066] Systems for cloning and expression of a polypeptide in a varietyof different host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli.

[0067] Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences, including promoter sequences,terminator fragments, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Vectors may beplasmids, viral e.g. phage, or phagemid, as appropriate. For furtherdetails see, for example, Molecular Cloning: a Laboratory Manual: 2ndedition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Current Protocols inMolecular Biology, Ausubel et al. eds., John Wiley & Sons, 1992.

[0068] Thus, a further aspect of the present invention provides a hostcell containing heterologous nucleic acid as disclosed herein.

[0069] The nucleic acid of the invention may be integrated into thegenome (e.g. chromosome) of the host cell. Integration may be promotedby inclusion of sequences which promote recombination with the genome,in accordance with standard techniques. The nucleic acid may be on anextra-chromosomal vector within the cell, or otherwise identifiablyheterologous or foreign to the cell.

[0070] A still further aspect provides a method which includesintroducing the nucleic acid into a host cell. The introduction, whichmay (particularly for in vitro introduction) be generally referred towithout limitation as “transformation”, may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage. As an alternative,direct injection of the nucleic acid could be employed.

[0071] Marker genes such as antibiotic resistance or sensitivity genesmay be used in identifying clones containing nucleic acid of interest,as is well known in the art.

[0072] The introduction may be followed by causing or allowingexpression from the nucleic acid, e.g. by culturing host cells (whichmay include cells actually transformed although more likely the cellswill be descendants of the transformed cells) under conditions forexpression of the gene, so that the encoded polypeptide (or peptide) isproduced. If the polypeptide is expressed coupled to an appropriatesignal leader peptide it may be secreted from the cell into the culturemedium. Following production by expression, a polypeptide or peptide maybe isolated and/or purified from the host cell and/or culture medium, asthe case may be, and subsequently used as desired, e.g. in theformulation of a composition which may include one or more additionalcomponents, such as a pharmaceutical composition which includes one ormore pharmaceutically acceptable excipiencs, vehicles or carriers (e.g.see below).

[0073] Introduction of nucleic acid encoding a peptidyl moleculeaccording to the present invention may take place in vivo by way of genetherapy, to disrupt or interfere with interaction between p21 and cyclinD1 and/or ckd4.

[0074] Thus, a host cell containing nucleic acid according to thepresent invention, e.g. as a result of introduction of the nucleic acidinto the cell or into an ancestor of the cell and/or genetic alterationof the sequence endogenous to the cell or ancestor (which introductionor alteration may take place in vivo or ex vivo), may be comprised (e.g.in the soma) within an organism which is an animal, particularly amammal, which may be human or non-human, such as rabbit, guinea pig,rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse,or which is a bird, such as a chicken. Genetically modified ortransgenic animals or birds comprising such a cell are also provided asfurther aspects of the present invention.

[0075] This may have a therapeutic aim. (Gene therapy is discussedbelow.) Also, the presence of a mutant, allele, derivative or variantsequence within cells of an organism, particularly when in place of ahomologous endogenous sequence, may allow the organism to be used as amodel in testing and/or studying substances which modulate activity ofthe encoded polypeptide in vitro or are otherwise indicated to be oftherapeutic potential. Conveniently, however, assays for such substancesmay be carried out in vitro, within host cells or in cell-free systems.

[0076] Suitable screening methods are conventional in the art. Theyinclude techniques such as radioimmunosassay, scintillation proximetryassay and ELISA methods. Suitably either the p21 protein or fragment orcyclinD1 and/or Cdk 4 or fragment, or an analogue, derivative, variantor functional mimetic thereof, is immobilised whereupon the other isapplied in the presence of the agents under test. In a scintillationproximetry assay a biotinylated protein fragment is bound tostreptavidin coated scintillant—impregnated beads (produced byAmersham). Binding of radiolabelled peptide is then measured bydetermination of radioactivity induced scintillation as the radioactivepeptide binds to the immobilized fragment. Agents which intercept thisare thus inhibitors of the interaction.

[0077] In one general aspect, the present invention provides an assaymethod for a substance with ability to disrupt interaction or bindingbetween p21 and cyclin D1 and/or Cdk4, the method including:

[0078] (a) bringing into contact a substance according to the inventionincluding a peptide fragment of p21 or a derivative, variant or analoguethereof as disclosed, a substance including the relevant fragment ofcyclin D1 and/or Cdk4 or a variant, derivative or analogue thereof, anda test compound, under conditions wherein, in the absence of the testcompound being an inhibitor of interaction or binding of saidsubstances, said substances interact or bind; and

[0079] (b) determining interaction or binding between said substances.

[0080] A test compound which disrupts, reduces, interferes with orwholly or partially abolishes binding or interaction between saidsubstances (e.g. including a p21 fragment and including a cyclin D1and/or Cdk 4 fragment), and which may modulate Cdk4 activity, may thusbe identified.

[0081] Another general aspect of the present invention provides an assaymethod for a substance able to bind the relevant region of p21 as thecase may be, the method including:

[0082] (a) bringing into contact a substance which includes a peptidefragment of p21 which interacts with cyclin D1 and/or Cdk 4 asdisclosed, or a variant, derivative or analogue thereof as disclosed,and a test compound; and

[0083] (b) determining binding between said substance and the testcompound.

[0084] A test compound found to bind to the relevant portion of p21 maybe tested for ability to disrupt p21 interaction or binding with cyclinD1 and/or Cdk 4 and/or ability to affect Cdk4 activity or other activitymediated by p21 as discussed already above.

[0085] Performance of an assay method according to the present inventionmay be followed by isolation and/or manufacture and/or use of acompound, substance or molecule which tests positive for ability tointerfere with interaction between p21 and cyclin D1 and/or Cdk4 and/orinhibit p21-mediated Cdk4 activity.

[0086] The precise format of an assay of the invention may be varied bythose of skill in the art using routine skill and knowledge. Forexample, interaction between substances may be studied in vitro bylabelling one with a detectable label and bringing it into contact withthe other which has been immobilised on a solid support. Suitabledetectable labels, especially for petidyl substances include³⁵S-methionine which may be incorporated into recombinantly producedpeptides and polypeptides. Recombinantly produced peptides andpolypeptides may also be expressed as a fusion protein containing anepitope which can be labelled with an antibody.

[0087] The protein which is immobilized on-a solid support may beimmobilized using an antibody against that protein bound to a solidsupport or via other technologies which are known per se. A preferred invitro interaction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to diminish theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

[0088] An assay according to the present invention may also take theform of an in vivo assay. The in vivo assay may be performed in a cellline such as a yeast strain or mammalian cell line in which the relevantpolypeptides or peptides are expressed from one or more vectorsintroduced into the cell.

[0089] The ability of a test compound to disrupt interaction or bindingbetween p21 and cyclin D1 and/or Cdk4 may be determined using aso-called two-hybid assay.

[0090] For example, a polypeptide or peptide containing a fragment ofp21 or cyclin D1/Cdk4 as the case may be, or a peptidyl analogue orvariant thereof as disclosed, may be fused to a DNA binding domain suchas that of the yeast transcription factor CAL 4. The GAL 4 transcriptionfactor includes two functional domains. These domains are the DNAbinding domain (GAL4 DBD) and the GAL4 transcriptional activation domain(GAL4TAD). By fusing one polypeptide or peptide to one of those domainsand another polypeptide or peptide to the respective counterpart, afunctional GAL 4 transcription factor is restored only when twopolypeptides or peptides of interest interact. Thus, interaction of thepolypeptides or peptides may be measured by the use of a reporter geneprobably linked to a GAL 4 DNA binding site which is capable ofactivating transcription of said reporter gene. This assay format isdescribed by Fields and Song, 1989, Nature 340; 245-246. This type ofassay format can be used in both mammalian cells and in yeast. Othercombinations of DNA binding domain and transcriptional activation domainare available in the art and may be preferred, such as the LexA DNAbinding domain and the VP60 transcriptional activation domain.

[0091] To take a Lex/VP60 two hybrid screen by way of example for thepurpose of illustration, yeast or mammalian cells may be transformedwith a reporter gene construction which expresses a selective markerprotein (e.g. encoding β-galactosidase or luciferase). The promoter ofthat gene is designed such that it contains binding site for the LexADNA-binding protein. Gene expression from that plasmid is usually verylow. Two more expression vectors may be transformed into the yeastcontaining the selectable marker expression plasmid, one containing thecoding sequence for the full length LexA gene linked to a multiplecloning site. This multiple cloning site is used to clone a gene ofinterest, i.e. encoding a p21 or cyclinD1/Cdk4 polypeptide or peptide inaccordance with the present invention, in frame on to the LexA codingregion. The second expression vector then contains the activation domainof the herpes simplex transactivator VP16 fused to a test peptidesequence or more preferably a library of sequences encoding peptideswith diverse e.g. random sequences. Those two plasmids facilitateexpression from the reporter construct containing this selectable markeronly when the LexA fusion construct interacts with a polypeptide orpeptide sequence derived from the peptide library.

[0092] A modification of this when looking for peptides or othersubstances which interfere with interaction between a p21 polypeptide orpeptide and an cyclin D1/Cdk 4 polypeptide or peptide, employs the p21or cyclin D1/Cdk4 polypeptide or peptide as a fusion with the LexA DNAbinding domain, and the counterpart cyclin D1/Cdk4 or p21 polypeptide orpeptide as a fusion with VP60, and involves a third expression cassette,which may be on a separate expression vector, from which a peptide or alibrary of peptides of diverse and/or random sequence may be expressed.A reduction in reporter gene expression (e.g. in the case ofβ-galactosidase a weakening of the blue colour) results from thepresence of a peptide which disrupts the p21/cyclinD1 and/or Cdk4interaction, which interaction is required for transcriptionalactivation of the β-galactosidase gene. Where a test substance is notpeptidyl and may not be expressed from encoding nucleic acid within asaid third expression cassette, a similar system may be employed withthe test substance supplied exogenously.

[0093] As noted, instead of using LexA and VP60, other similarcombinations of proteins which together form a functionaltranscriptional activator may be used, such as the GAL4 DNA bindingdomain and the GAL4 transcriptional activation domain.

[0094] When performing a two hybrid assay to look for substances whichinterfere with the interaction between two polypeptides or peptides itmay be preferred to use mammalian cells instead of yeast cells. The sameprinciples apply and appropriate methods are well known to those skilledin the art.

[0095] The amount of test substance or compound which may be added to anassay of the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 nM concentrations of putative inhibitor compound may be used, forexample from 0.1 to 10 nM. Greater concentrations may be used when apeptide is the test substance.

[0096] Compounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants whichcontain several characterised or uncharacterised components may also beused.

[0097] Antibodies directed to the site of interaction in either proteinform a further class of putative inhibitor compounds. Candidateinhibitor antibodies may be characterised and their binding regionsdetermined to provide single chain antibodies and fragments thereofwhich are responsible for disrupting the interaction.

[0098] Antibodies may be obtained using techniques which are standard inthe art. Methods of producing antibodies include immunising a mammal(e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the proteinor a fragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al., 1992, Nature 357: 80-82). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

[0099] As an alternative or supplement to immunising a mammal with apeptide, an antibody specific for a protein may be obtained from arecombinantly produced library of expressed immunoglobulin variabledomains, e.g. using lambda bacteriophage or filamentous bacteriophagewhich display functional immunoglobulin binding domains on theirsurfaces; for instance see WO92/01047. The library may be naive, that isconstructed from sequences obtained from an organism which has not beenimmunised with any of the proteins (or fragments), or may be oneconstructed using sequences obtained from an organism which has beenexposed to the antigen of interest.

[0100] Antibodies according to the present invention may be modified ina number of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimicks that of an antibody enablingit to bind an antigen or epitope.

[0101] Example antibody fragments, capable of binding an antigen orother binding partner are the Fab fragment consisting of the VL, VH, C1and CH1 domains; the Fd fragment consisting of the VH and CH1 domains;the Fv fragment consisting of the VL and VH domains of a single arm ofan antibody; the dAb fragment which consists of a VH domain; isolatedCDR regions and F(ab′)₂ fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

[0102] A hybridoma producing a monoclonal antibody according to thepresent invention may be subject to genetic mutation or other changes.It will further be understood by those skilled in the art that amonoclonal antibody can be subjected to the techniques of recombinantDNA technology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP184187A, GB 2188638A orEP-A-0239400. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

[0103] Hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells, eukaryotic or prokaryotic, containing nucleic acid encodingantibodies (including antibody fragments) and capable of theirexpression. The invention also provides methods of production of theantibodies including growing a cell capable of producing the antibodyunder conditions in which the antibody is produced, and preferablysecreted.

[0104] The reactivities of antibodies on a sample may be determined byany appropriate means. Tagging with individual reporter molecules is onepossibility. The reporter molecules may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

[0105] One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

[0106] Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronicallv detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

[0107] The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

[0108] Antibodies may also be used in purifying and/or isolating apolypeptide or peptide according to the present invention, for instancefollowing production of the polypeptide or peptide by expression fromencoding nucleic acid therefor. Antibodies may be useful in atherapeutic context (which may include prophylaxis) to disruptp21/cyclin D1/Cdk4 interaction with a view to inhibiting Cdk4 activityand so cellular proliferation. Antibodies can for instance bemicro-injected into cells, e.g. at a tumour site.

[0109] Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size and charge characteristics.

[0110] A compound found to have the ability to affect Cdk4 activity hastherapeutic potential in anti-tumour treatment, and may be used incombination with any other anti-tumour compound. In such a case, theassay of the invention, when conducted in vivo, need not measure thedegree of inhibition of binding or of modulation of Cdk4 activity causedby the compound being tested. Instead the effect on tumorigenicityand/or cell viability may be measured. It may be that such a modifiedassay is run in parallel with or subsequent to the main assay of theinvention in order to confirm that any effect on tumorigenicity orand/or cell viability is as a result of the inhibition of binding orinteraction between p21 and cyclin D1/Cdk 4 caused by said inhibitorcompound and not merely a general toxic effect.

[0111] Following identification of a substance or agent which modulatesor affects Cdk4 activity, the substance or agent may be investigatedfurther. Furthermore, it may be manufactured and/or used in preparation,i.e. manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

[0112] As noted, the agent may be peptidyl, e.g. a peptide whichincludes a sequence as recited above, or may be a functional analogue ofsuch a peptide.

[0113] As used herein, the expression “functional analogue” relates topeptide variants or organic compounds having the same functionalactivity as the peptide in question, which may interfere with thebinding between p21 and cyclin D1/Cdk4. Examples of such analoguesinclude chemical compounds which are modelled to resemble the threedimensional structure of the p21 or cyclin D1 /Ckd4 domain in thecontact area, and in particular the arrangement of the key amino acidresidues identified above as they appear in human p21.

[0114] In a further aspect, the present invention provides the use ofthe above substances in methods of designing or screening for mimeticsof the substances.

[0115] Accordingly, the present invention or provides a method ofdesigning mimetics of p21^(WAF1) having the biological activity of Cdk4binding or inhibition, the activity of allosteric inhibition of Cdk4and/or the activity of cyclin D1 binding, said method comprising:

[0116] (i) analysing a substance having the biological activity todetermine the amino acid residues essential and important for theactivity to define a pharmacophore; and,

[0117] (ii) modelling the pharmacophore to design and/or screencandidate mimetics having the biological activity.

[0118] Suitable modelling techniques are known in the art. This includesthe design of so-called “mimetics”>which involves the study of thefunctional interactions fluorogenic oligonucleotide the molecules andthe design of compounds which contain functional groups arranged in sucha manner that they could reproduced those interactions.

[0119] The designing of mimetics to a known pharmaceutically activecompound is a known approach to the development of pharmaceuticals basedon a “lead” compound. This might be desirable where the active compoundis difficult or expensive to synthesise or where it is unsuitable for aparticular method of administration, e.g. peptides are not well suitedas active agents for oral compositions as they tend to be quicklydegraded by proteases in the alimentary canal. Mimetic design, synthesisand testing may be used to avoid randomly screening large number ofmolecules for a target property.

[0120] There are several steps commonly taken in the design of a mimeticfrom a compound having a given target property. Firstly, the particularparts of the compound that are critical and/or important in determiningthe target property are determined. In the case of a peptide, this canbe done by systematically varying the amino acid residues in thepeptide, e.g. by substituting each residue in turn. These parts orresidues constituting the active region of the compound are known as its“pharmacophore”.

[0121] Once the pharmacophore has been found, its structure is modelledto according its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.spectroscopic techniques, X-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

[0122] In a variant of this approach, the three-dimensional structure ofthe ligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

[0123] A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore can be grafted. The template molecule andthe chemical groups grafted on to it can conveniently be selected sothat the mimetic is easy to synthesise, is likely to bepharmacologically acceptable, and does not degrade in vivo, whileretaining the biological activity of the lead compound. The mimetic ormimetics found by this approach can then be screened to see whether theyhave the target property, or to what extent they exhibit it. Furtheroptimisation or modification can then be carried out to arrive at one ormore final mimetics for in vivo or clinical testing.

[0124] The mimetic or mimetics found by this approach can then bescreened to see whether they have the target property, or to what extentthey exhibit it. Further optimisation or modification can then becarried out to arrive at one or more final mimetics for in vivo orclinical testing.

[0125] Mimetics of this type together with their use in therapy form afurther aspect of the invention.

[0126] The present invention further provides the use of a peptide whichincludes a sequence as disclosed, or a derivative, active portion,analogue, variant or mimetic, thereof able to bind Cdk4 and/or inhibitCdk4 activity, in screening for a substance able to bind Cdk4 and/orinhibit Cdk4 activity. Generally, an inhibitor according to the presentinvention is provided in an isolated and/or purified form, i.e.substantially pure. This may include being in a composition where itrepresents at least about 90% active ingredient, more preferably atleast about 95%, more preferably at least about 98%. Such a compositionmay, however, include inert carrier materials or other pharmaceuticallyand physiologicaly acceptable excipients. As noted below, a compositionaccording to the present invention may include in addition to aninhibitor compound as disclosed, one or more other molecules oftherapeutic use, such as an anti-tumour agent.

[0127] The present invention extends in various aspects not only to asubstance identified as a modulator of p21 and cyclin D1/Ckd4interaction and/or Cdk4-mediated RB phosphorylation or other substratesof Cdk4 or other p21-mediated activity, property or pathway inaccordance with what is disclosed herein, but also a pharmaceuticalcomposition, medicament, drug or other composition comprising such asubstance, a method comprising administration of such a composition to apatient, e.g. for anti-tumour or other anti-proliferative treatment,which may include preventative treatment, use of such a substance inmanufacture of a composition for administration, e.g. for anti-tumour orother anti-proliferative treatment, and a method of making apharmaceutical composition comprising admixing such a substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

[0128] A substance according to the present invention such as aninhibitor of p21 and cyclin D1 and/or Cdk4 interaction or binding may beprovided for use in a method of treatment of the human or animal body bytherapy which affects Cdk 4 activity or other p21-mediated activity incells, e.g. tumour cells.

[0129] Thus the invention further provides a method of modulating Cdk4activity, or other p2′-mediated activity in a cell, which includesadministering an agent which inhibits or blocks the binding of p21 tocyclin D1 and/or Cdk4 protein, such a method being useful in treatmentof cancer or other diseases or disorders including malignancies whereinhibition of cellular growth and/or proliferation is desirable.

[0130] The invention further provides a method of treating tumours whichincludes administering to a patient an agent which interferes with thebinding of p21 to cyclin D1 and/or Cdk4.

[0131] Whether it is a polypeptide, antibody, peptide, nucleic acidmolecule, small molecule, mimetic or other pharmaceutically usefulcompound according to the present invention that is to be given to anindividual, administration is preferably in a “prophylacticallyeffective amount” or a “therapeutically effective amount” (as the casemay be, although prophylaxis may be considered therapy), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practioners and other medical doctors.

[0132] A composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated.

[0133] Pharmaceutical compositions according to the present invention,and for use in accordance with the present invention, may include, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. cutaneous,subcutaneous or intravenous.

[0134] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0135] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilisers, buffers, antioxidants and/or other additives may beincluded, as required.

[0136] Examples of techniques and protocols mentioned above can be foundin Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed),1980.

[0137] The agent may be administered in a localised manner to a tumoursite or other desired site or may be delivered in a manner in which ittargets tumour or other cells.

[0138] Targeting therapies may be used to deliver the active agent morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons, for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

[0139] Instead of administering these agents directly, they may beproduced in the target cells by expression from an encoding geneintroduced into the cells, eg in a viral vector (a variant of the VDEPTtechnique—see below). The vector may targeted to the specific cells tobe treated, or it may contain regulatory elements which are switched onmore or less selectively by the target cells.

[0140] The agent may be administered in a precursor form, for conversionto the active form by an activating agent produced in, or targeted to,the cells to be treated. This type of approach is sometimes known asADEPT or VDEPT, the former involving targeting the activating agent tothe cells by conjugation to a cell-specific antibody, while the latterinvolves producing the activating agent, e.g. an enzyme, in a vector byexpression from encoding DNA in a viral vector (see for example,EP-A-415731 and WO 90/07936).

[0141] A composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated, such as cancer, virus infection or anyother condition in which a p21-mediated effect is desirable.

[0142] Nucleic acid according to the present invention, encoding apolypeptide or peptide able to interfere with p21 and cyclin D1 and/orCdk 4 interaction or binding and/or induce or modulate Cdk4 activity orother p21-mediated cellular pathway or function, may be used in methodsof gene therapy, for instance in treatment of individuals with the aimof preventing or curing (wholly or partially) a tumour e.g. in cancer,or other disorder involving loss of proper regulation of the cell-cycleand/or cell growth, or other disorder in which specific cell death isdesirable, such as in certain viral infections.

[0143] Vectors such as viral vectors have been used in the prior art tointroduce nucleic acid into a wide variety of different target cells.Typically the vectors are exposed to the target cells so thattransfection can take place in a sufficient proportion of the cells toprovide a useful therapeutic or prophylactic effect from the expressionof the desired polypeptide. The transfected nucleic acid may bepermanently incorporated into the genome of each of the targeted tumourcells, providing long lasting effect, or alternatively the treatment mayhave to be repeated periodically.

[0144] A variety of vectors, both viral vectors and plasmid vectors, areknown in the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. Inparticular, a number of viruses have been used as gene transfer vectors,including papovaviruses, such as SV40, vaccinia virus, herpesviruses,including HSV and EBV, and retroviruses. Many gene therapy protocols inthe prior art have used disabled murine retroviruses.

[0145] As an alternative to the use of viral vectors other known methodsof introducing nucleic acid into cells includes electroporation, calciumphosphate co-precipitation, mechanical techniques such asmicroinjection, transfer mediated by liposomes and direct DNA uptake andreceptor-mediated DNA transfer.

[0146] Receptor-mediated gene transfer, in which the nucleic acid islinked to a protein ligand via polylysine, with the ligand beingspecific for a receptor present on the surface of the target cells, isan example of a technique for specifically targeting nucleic acid toparticular cells.

[0147] A polypeptide, peptide or other substance able to interfere withthe interaction of the relevant polypeptide, peptide or other substanceas disclosed herein, or a nucleic acid molecule encoding a peptidyl suchmolecule, may be provided in a kit, e.g. sealed in a suitable containerwhich protects its contents from the external environment. Such a kitmay include instructions for use.

[0148] Various further aspects and embodiments of the present inventionwill be apparent to those skilled in the art in view of the presentdisclosure. Certain aspects and embodiments of the invention will now beillustrated by way of example and with reference to the followingfigures:

[0149]FIG. 1. The Ability of Peptides from p21^(WAF1) to Interact withCdk4 and Cyclin D1.

[0150]FIG. 1a: a list of the peptides 1-11 based on the sequence ofp21^(WAF1). FIG. 1b: The p21^(WAF1) peptides were bound tostreptavidin-agarose beads and added to reticulocyte lysates containingeither Cdk4 or cyclin D1 labelled with [³⁵S] methionine. After extensivewashing bound proteins were analysed using SDS-PAGE followed byautoradiography. The bands were quantified using a Bio-Imager and WholeBand Analysis Software (Millipore). The results are representative of 3such experiments. “x” indicates beads without peptide.

[0151]FIG. 2. Addition of p21^(WAF1) Based Peptides to Cyclin D1-Cdk4Phosphorylation Assays.

[0152] Cyclin D1-Cdk4 assays were carried out in vitro using lysatesfrom Sf9 insect cell following co-infection with Cdk4 and cyclin D1baculovirus constructs and GST-Rb as the substrate. p₂₁ ^(WAF1) peptideswere added to the assays at a concentration of 17 μM and the effect onCdk4 activity was assessed by SDS-PAGE and autoradiography. The figureshows quantification of the autoradiograph using bio-imaging, relativebinding is expressed in terms of Cdk4 activity in the absence ofpeptide. The data are representative of 4 experiments. “x” indicates noaddition of peptide.

[0153]FIG. 3. Quantification of Peptide Inhibition.

[0154] Peptides 4, 8, 2 and 10 were added to cyclin D1-Cdk4 assays atvarious concentrations between 0.01-34 μM. The figure gives a plot ofactivity (%) relative to Cdk4 activity measured in the absence ofpeptide against peptide concentration and the I_(0.5) for each peptide.The data represent the mean of 3 experiments.

[0155]FIG. 4. Peptides 2 or 10 are Not Substrates for Cyclin D1-Cdk4.

[0156] The figure shows the results of phosphorylation assays usingpeptides 2, 4 & 10.

[0157]FIG. 5. Size Scan of Peptide 10.

[0158] The figure shows the sequences of a series of peptides based onpeptide 10 designed to find the minimal inhibitory domain. The boxedresidues represent the minimal inhibitory domain. The peptides wareadded to cyclin D1-Cdk4 assays and analysed by SDS-PAGE andautoradiography.

[0159]FIG. 6. Alanine Scan Mutations of Peptide 10.

[0160] In order to pinpoint residues that were critical for theinhibition of Cdk4 by peptide 10 a series of point mutations wereconstructed in which each residue was sequentially changed to alanine.The peptides were added to cyclin D1-Cdk4 assays and the results wereanalysed by SDS-PAGE and autoradiography then quantified using aBio-Imager. The results are expressed relative to Cdk4 activity in theabsence of peptide and are representative of 3 experiments. Havingidentified the critical residues we then synthesised an untagged eightamino acid peptide which contained the R, L and F (KRRLIFS) anddetermined the phosphorylation of GST-Rb by cyclin D1-Cdk4 in thepresence of increasing concentrations of this truncated peptide.

[0161]FIG. 7. Comparison of Inhibitory Peptides with Full Lengthp21^(WAF1) Protein.

[0162] This shows concentration curves for peptide 10, D to A mutantpeptide 10, a p16INK4 derived peptide (Fahraeus et al., 1996) and fulllength his-p21^(WAF1) determined using the cyclin D1-Cdk4 assay analysedby SDS-PAGE, autoradiography and bio-imaging and for the I_(0.5) of eachinhibitor. The results are the mean of 3 such experiments.

[0163]FIG. 8. Binding and Inhibitory Domains of p₂₁ ^(WAF1).

[0164] The hatched residues show the regions of p21^(WAF1) identified inthis study as being important for cyclin D1 and Cdk4 binding, and Cdk4inhibition in the N-terminal domain, as well as a novel inhibitorydomain in the C-terminus of p21^(WAF1) The residues found to beimportant for the interaction of p21^(WAF1) with PCNA (Warbrick et al.,1995) are shown in black. In addition, the smallest portion ofp21^(WAF1) that was found to inhibit CDK activity in vitro (Luo et al.,1995) prior to the present study is indicated.

[0165]FIG. 9. Introduction of p₂₁ ^(WAF1) Based Peptides Into Cells

[0166] A series of synthetic peptides based on the sequence of peptide10 (Peptides I, II and III, shown in FIG. 9a) were synthesised withcarrier peptide (shaded sequences). The underlined residue in Peptide-1is the M to A mutation which prevents PCNA binding. The peptides wereadded to proliferating HaCaT cells, grown in DMEM plus 10% FCS. Thecells were incubated for 24 hours pulse labelled during with 15 μM BrdU,fixed and then analysed by FACS. The G₁-, S— and G₂-phase distributionsfor untreated cells, Peptide-I at 25 μM, Peptide-II at 50 μM andPeptide-III at 25 μM, were determined. FIG. 9b shows the datarepresented as the % of cells in each phase compared to the total numberof cells counted. The results of similar experiments using MCF7 and MRCScells are shown in FIG. 9c, which shows the percentage of cells in eachphase of the cell cycle in the absence and presence of peptide I.

[0167] In a separate experiment DMEM+10%, FCS alone or DMEM+10% FCScontaining either 25 μM Peptide-I or 50 μM Peptide-II, was added toHaCaT cells than had been starved for 72 hours. Samples were taken atthe times shown and analysed by SDS-PAGE/Western blot stained for pRb.pRb represents hypophosphorylated Rb protein and pRb* refers tohyperphosphorylated Rb protein. It should be pointed out that equalamounts of total protein were loaded per lane and that the antibodyappears to preferentially recognise phosphorylated forms of the Rbprotein.

[0168]FIG. 10. Inhibition of Cyclin-Cdk4 Activity Using Derivatives ofPeptide 2.

[0169] The figure shows the degree of inhibition of cyclin D1-Cdk4activity using pRb as a substrate, by peptide 2 (2 on the figure) andalanine scan mutations of the peptide (each residue being sequentiallymutated to alanine) Activity is given relative to uninhibited activity.(No is short for no added peptide.) Peptides were present at aconcentration of 10 μM. A similar pattern is seen when binding ofpeptide 2 mutants to cyclin D1 expressed in reticulocyte lysates.

[0170] Important residues for the binding and inhibition are the twoarginine residues (R) and the phenylalanine (F) with the lysine (K) andproline (P) also contributing. This is different from residuesidentified as being critical for interaction of full length protein withcyclin D1, as these studies pick out the LFG motif as being mostimportant for activity.

[0171] Experimental Procedures

[0172] Peptides

[0173] All peptides were synthesised by Chiron Mimotopes, PeptideSystems (Clayton, Australia). Each peptide had a Biotin-SGSG spacer atthe C-terminus and a free N-terminus. The peptides were dissolved inDMSO at approximately 5 mg/ml and we then determined their concentrationprecisely by amino acid analysis (Smythe et al., 1988). In addition thepurity of the peptides was estimated using mass spectrometry. Positiveion electrospray mass spectrometry was performed on a triple-quadruplemass spectrometer (V. G. Quattro) in (50/50/0.1)water/acetonitrile/formic acid.

[0174] Proteins

[0175] Cyclins and CDKs—Cdk4 and cyclin D1, Cdk2 and cyclin E and Cdc2and cyclin B were co-expressed in Sf9 insect cells infected with theappropriate baculovirus constructs. The cells were harvested two daysafter infection by low speed centrifugation and the pellet was lysed inan equal volume of 10 mM Hepes, pH 7.4 containing: 10 mM NaCl, 1 mMEDTA, and 0.1 mM phenylmethane sulphonyl fluoride, 2 mM DTT andcentrifuged at 14000×g for 15 min. The supernatant was removed,aliquoted and immediately frozen in liquid nitrogen. Thawed lysate wasused only once and was never refrozen. Labelled Cdk4 and cyclin D1 wereproduced by translation in the presence of [³⁵S] methionine using arabbit reticulocyte lysate in vitro translation kit (Promega) His-taggedp21^(WAF1) Human p21^(WAF1) was expressed in E. coli using a PETexpression vector. The soluble p21^(WAF1) protein fraction was purifiedusing a nickel chelating column, following the manufacturersinstructions (Pharmacia). The eluted protein peak was dialysed against25 mM Hepes, pH 7.4, containing: 0.1 mM EDTA, 1 mm benzamidine, 0.01%Triton X-100, and 0.1 mM phenylmethane sulphonyl fluoride, concentratedand applied to a Superose 12 gel-filtration column (Phamacia)equilibrated in the above buffer. Fractions containing p21^(WAF1) weredetected by Western blot using the p21^(WAF1) specific monoclonalantibody Ab-1 (oncogene Sciences), concentrated to 200 μg/ml and storedat −70° C.

[0176] GST-Rb—An E. coli expression construct containing thehyperphosphorylation domain of pRb (amino acids 773-924) was purified ona glutathione-Sepharose column according to the manufacturersinstructions (Pharmacia).

[0177] Peptide Precipitation of Cdk4 and Cyclin D1

[0178] A 20 amino acid peptide library, that spanned the entire sequenceof p21^(WAF1) (FIG. 1), was screened for Cdk4/cyclin D1 interactingpeptides. Peptide (1.5 μg) was diluted in 100 μl of PBS and incubatedwith 10 μl of packed streptavidin-agarose beads (Sigma) for 1 h at roomtemperature. Unbound peptide was removed by extensive washing with PBSand the beads, plus bound peptide, were incubated for 1 h at 4° C. withreticulocyte lysate containing either Cdk4 or cyclin D1 labelled with[³⁵S] methionine. The beads were washer three times with 1.25×PBScontaining 0.2% Triton X-100 and boiled in the presence of 0.125 MTris-HCl, pH 6.8 containing: 4% (w/v) SDS, 20% (v/v) glycerol and 200 mMDTT. The bound protein was analysed by SDS-PAGE followed byauto-radiography and quantification of the ³⁵S-labelled protein using aBio-Imager and Whole Band Analysis Software (Millipore).

[0179] Enzyme Assays

[0180] Phosphorylation of GST-Rb—Cdk4 activity was measured using thecyclin D1-Cdk4 containing insect cell lysate described above. Extract (1μl) was added to a final reaction volume of 10/1, containing: 50 mMHepes, pH 7.4, 10 mM MgCl2, 2.5 mM EGTA, 1 mM DTT, 10 mMβ-glycerophosphate, 1 mM NaF, 10 mM PKI, 50 μM ATP containing [³²P] ATP(1 000 cpm/pMol) and 0.5 μg GST-Rb. The assays were started by theaddition of the GST-Rb substrate, incubated at 30° C. for 10 min (theincorporation of ³² P into GST-Rb was linear over 1S-20 min) andterminated by adding SDS-PAGE sample buffer and heating at 95° C. for 4min. The samples were analysed by SDS-PAGE on 12% gels followed byauto-radiography and quantification using a Bio-Imager.

[0181] Peptide Phosphorylation

[0182] The biotinylated peptides (1 μg) were incubated for 30 min at 30°C. in a final volume of 20 μl containing: 50 mM Hepes, pH 7.4, 10 mMMgCl₂, 2.5 mM EGTA, 1 mM DTT, 10 mM β-glycerophosphate, 1 mM NaF, 10 μMPKI, 50 μM ATP containing [³²P]ATP (6000 cpm/pMol) and either 1 μl ofcyclin D1-Cdk4 insect cell lysate, 1 μl of uninfected insect cell lysateor 0.02 mU of protein kinase C plus 0.5 mM CaCl₂, 100 mg/ml phosphatidylserine and 20 mg/ml diacylglycerol. The reactions were stopped byheating at 60° C. for 5 min and streptavidin agarose beads were added(10 μl packed cell volume washed with 3×PBS) and incubated with shakingat 4° C. for 30 min. The beads were washed extensively with PBScontaining 3% (v/v) Tween-20 and the incorporation of radioactivity intothe peptides was determined by Cerenkov counting.

[0183] Cell Cycle Measurements

[0184] Carrier linked peptides were designed for delivery intoproliferating HaCaT cells (see FIG. 9). Cells were seeded on 30 mmculture plates and grown to 50's confluency in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10′ (v/v) foetal calf serum(FCS). Peptides were added to the medium and the cells were incubatedfor 24 hours. During the last 30 minutes of the incubation the cellswere pulse labelled in the presence of 15 μM BrdU. The cells weretrypsinised, fixed in absolute alcohol and prepared for FACS analysisusing a single laser flow cytometer (Becton-Dickinson, FACScan) aspreviously described (Renzing et al, 1996).

[0185] pRb Phosphorylation in HaCaT Cells

[0186] HaCaT cells were seeded on 30 mm culture plates at 25% confluencyin DMEM with 10% FCS. The FCS was withdrawn after 24 hours and the cellswere starved for 72 hours. At the end of this period the medium wassupplemented with 10% FCS and carrier linked peptides. Samples weretaken over a 24 hour time course and the cells were lysed in RIPA buffer(50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 1.0% (v/v) NP-40, 0.5%(w/v) DOC, 0.1%. (w/v) SDS, 1 mM PMSF, 0.1 mg/ml aprotinin and 0.5 mg/mlleupeptin) for 30 minutes at 4° C. The phosphorylation statues of pRbwas determined by Western blot analysis, as previously described(Fahraeus et al, 1996) except that the blot was probed with a pRbpolyclonal antibody (C-15, Santa Cruz).

[0187] Results

[0188] Peptide-Binding Assay for Cyclin D1 and Cdk1

[0189] Using a series of synthetic peptides that span the entiresequence of p21^(WAF1) (FIG. 1), we determined whether these peptidescould mimic full length p21^(WAF1) protein by forming a stable complexwith either cyclin D1 or Cdk4. If peptide-binding mimetics of p21^(WAF1)protein could be identified, then this would assist in identifying theminimal binding motif of p21^(WAF1) protein required for cyclin D1-Cdk4holoenzyme inhibition and whether p21^(WAF1) was targeting the cyclin orthe kinase subunit. This would also define a system for using smallpeptides to study p21^(WAF1) protein reaction mechanism and to designmimetic drugs.

[0190] The peptide-binding assay involved quantifying the amount of³⁵S-labelled cyclin D1 or Cdk4 which bound specifically to biotinylatedpeptides that were captured on streptavidin coated agarose beads. Thepeptide-coated beads were added to extracts containing either³⁵S-labelled cyclin D1 or Cdk4 translated in vitro, the beads werewashed extensively to remove unbound protein, and the bound cyclin D1 orCdk4 was quantified by SDS-PAGE followed by auto-radiography andbio-imaging. This is referred to below as a peptide precipitation assayand has been used previously to demonstrate evolutionary conservation ofp21^(WAF1) binding to PCNA (Ball and Lane, 1996).

[0191] A Small Peptide Derived from Amino Acids 46-65 in the N-TerminalDomain of p21^(WAF1) Binds Directly to Cdk4

[0192] Using the peptide-precipitation assay, peptide 4 (from theN-terminal domain of p21^(WAF1)) bound specifically to Cdk4, but not tocyclin D1 (FIG. 1). This interaction is physiologically important, sincethe CDK interacting domain of the p21^(WAF1) protein has previously beenproposed to localise to the N-terminal domain of the molecule (Chen etal., 1995; Harper et al., 1995; Luo et al., 1995). More specifically,deletions (Nakanishi et al., 1995a) or mutations (Goubin and Ducommun,1995) in the region of amino acids 45-71 compromise the ability of fulllength p21^(WAF1) to interact with Cdk2. Whether this loss of p21^(WAF1)binding function is due to, (i) mutation/deletion of residues directlyinvolved in CDK binding, or (ii) mutations/deletion inducedconformational alterations in p21^(WAF1) that prevent stable binding toCDK, has not been demonstrated. Here we show unequivocally that residues46-65 are directly involved in the binding of p21 to Cdk4 and that alonethey are capable of forming a stable complex with Cdk4, in the absenceof cyclin D1 . Thus, providing direct evidence that the N-terminus ofp21^(WAF1) does contain a kinase binding domain.

[0193] A Small Peptide Derived from Amino Acids 16-35 in the N-Terminusof p₂₁ ^(WAF1) Binds Directly to Cyclin D1

[0194] We were also able to define a second and distinct N-terminalinteraction site on the p21^(WAF1) protein; in this case a region ofp21^(WAF1) which is capable of binding to cyclin D1, but not to Cdk4(FIG. 1). Peptide 2 comprises amino acids 16-35 of p21^(WAF1) and lieswithin the Eminimum region required for DNA synthesis inhibition invivo, which is located between residues 17-71 (Nakanishi et al., 1995a).Our results might explain an apparent contradiction encountered byNakanishi et al. (1995a) who found that N-terminal mutations inp21^(WAF1) protein which are outside the CDK interacting domain,although insufficient to prevent binding to the kinase, were sufficientto prevent p21^(WAF1) from acting as a growth suppressor whentransfected into proliferating cells. Specifically, the direct peptidebinding data (FIG. 1) leads us to suggest that an N-terminal motif inthe p₂₁ ^(WAF1) protein, that mediates cyclin D1 binding, could be anessential step in the mechanism through which p21^(WAF1) proteinfunctions as a growth suppressor.

[0195] A Novel Cyclin D1-Cdk4 Binding Motif Resides in the C-Terminus ofthe p21^(WAF1) Protein

[0196] The specificity of the peptide-precipitation assay in definingthe domain of p21^(WAF1) protein required for binding to either thecyclin D1 or Cdk4 (FIG. 1), indicated that using peptides to studypotential interactions between p₂₁ ^(WAF1) and cyclin-CDK complexeswould prove to be very informative. We were intrigued however, by thefinding that peptides from the C-terminus of the p₂₁ ^(WAF1) protein(peptides 10 and 11) could form stable complexes with both Cdk4 andcyclin D1 (FIG. 1), as peptide 10 is equivalent to the p21PBP peptidedescribed by Warbrick et al. (1995) as representing the region ofp21^(WAF1) which binds to the replication/repair protein PCNA. We cannot rule out the possibility that endogenous cyclin or CDK present inthe reticulocyte lysate could bind to the labelled human protein forminga bridge to the peptide. However, as peptide 2 and peptide 4 precipitateeither cyclin D1 or Cdk4, respectively, this seems unlikely. Theseresults suggest that the p₂₁ ^(WAF1) protein may interact with both PCNAand cyclin-CDK complexes through the same binding motif. Peptide 11however, binds to both Cdk4 and cyclin D1 but not to PCNA (FIG. 1)(Warbrick et al., 1995; Ball and Lane, 1996); uncoupling the PCNAbinding site from the cyclin/CDK binding motif in the C-terminus ofp21^(WAF1).

[0197] Given that we had identified three distinct motifs from thep21^(WAF1) protein which bind specifically to cyclin D1 and/or Cdk4, wethen examined whether they mimicked p21^(WAF1) protein by inhibitingkinase activity.

[0198] The Cyclin D1 Binding Peptide from the N-Terminal Domain ofp21^(WAF1) nd the Cyclin/CDK Binding Peptide from the C-Terminus ofp21^(WAF1) Inhibits the Activity of Cdk4

[0199] In order to determine if any of the p21^(WAF1) peptides possessedCdk4 inhibitory activity we tested, independently, their ability toprevent pRb phosphorylation during cyclin D1-Cdk4 assays in vitro (FIG.2). Peptides 2, 8, 10, and 11 inhibited cyclin D1-Cdk4 activity whenadded to the assay at 17 μM, whereas buffer alone and the remainingpeptides had no dramatic affect on Cdk4 activity. The cyclin D1 bindingpeptide (peptide 2) inhibited the kinase activity by approximately 80%and peptides 10 and 11, which bound both Cdk4 and cyclin D1, completelyinhibited enzyme activity at this concentration. Thus, there is acorrelation between the ability of the peptides to bind to Cdk4 and/orcyclin D1 and to inhibit Cdk 4 kinase activity.

[0200] However, this correlation breaks down in the case ofkinase-binding peptide 4. This peptide maps to the CDK interaction site(FIG. 8; Goubin and Ducommun, 1995; Nakanishi et al., 1995a) and therehas been speculation that a peptide from this domain, capable ofinteracting with CDK, would mimic full-length p21^(WAF1) inhibitoryactivity, and would therefore provide a model for the design of novelmolecules that could arrest cell cycle progression by inhibiting the G1cyclin-CDKs. Although of high affinity for Cdk4, peptide 4 had noinhibitory activity when added to cyclin D1-Cdk4 assays atconcentrations of up to 35 μM. Our data from both p21^(WAF1)-peptidebinding data and inhibitory properties, therefore pinpoints two novelsmall domains of the p21^(WAF1) protein as potential candidates forsmall molecular weight mimetics; an N-terminal motif from amino acids16-35 (peptide 2) and a C-terminal motif from amino acids 141-10(peptide 10).

[0201] It is thus possible that peptide 4 could block p21 binding,preventing its activity as an inhibitor. Thus cells treated with peptide4 may be expected to continue to proliferate even in the presence ofcompeting signals which would normally mediate cell cycle arrest orapoptosis. Thus peptide 4 may be used to reversibly immortalise cells,by supplying to the peptide to the cells. This provides a further toolin investigating the cellular mechanisms for control of the cell cycleand may also be useful in combatting cell loss in conditions associatedwith loss of cells, such as in AIDS or degenerative conditions includingMS, dementia, or in muscle degenerative conditions such as musculardystropy (MD) including Duchenne MD.

[0202] The C-Terminal p21^(WAF1) Peptide is a More Potent Inhibitor ofCdk4 Kinase Activity than the N-Terminal Cyclin D1-Binding Peptide

[0203] We carried out more detailed studies to determine the I_(0.5) forpeptides 2, 8, and 10, using peptide 4 as a negative control (FIG. 3).We found that peptide 10 (and peptide 11; data not shown) was a potentinhibitor of Cdk4 activity with an I_(0.5) of 0.1 μM, peptide 2, wasalso a good inhibitor with an I_(0.5) of 2 μM. Peptide 8 gave only weakinhibition and relatively high concentrations of peptide were requiredto approach 50% inhibition. These data support the possibility of usingpeptide 2 or peptide 10 to mimic the CDK inhibitory activity of the fulllength p21^(WAF1) protein.

[0204] p21^(WAF1) Protein and Inhibitory Peptides Compete for the SameBinding Site on Cdk4 Kinase

[0205] In order to determine if the Cdk4 inhibitory peptides, 2 and 10,were acting at sites on Cdk4 and cyclin D1 that were also employed byp21^(WAF1), we carried out peptide precipitation assays in the presenceand absence of full length purified his-p21^(WAF1) to find out if itcompeted with the peptides for binding.

[0206] The ability of p21^(WAF1) to interfere with peptide 2 (A) andpeptide 10 (B & C) binding to Cdk4 and/or cyclin D1 was determined bycarrying out the peptide precipitation assay from reticulocyte lysatesin the presence of 0, 0.5, 2 μg of p21^(WAF1).

[0207] The data suggest that binding of p21^(WAF1) protein to cyclin D1and Cdk4 prevents binding of both peptide 2 and peptide 10. These dataare open to two interpretations, (i) the peptides could be competing forbinding at the same site as p21^(WAF1), or (ii) binding of eitherp21^(WAF1) or peptide could cause a conformational change in the cyclinor CDK preventing further binding. It is not clear from theseexperiments whether peptides 2 and 10 are acting at the same site(s).However the difference in the peptide precipitation data, indicates thatat least one of the sites is unique, as peptide 10 can precipitate bothCdk4 and cyclin D1, whereas, peptide 2 can only precipitate cyclin D1.

[0208] Data to support the hypothesis that peptide 10 and p₂₁ ^(WAF1)protein compete for the same binding site, during kinase inhibition,employs the use of a peptide 10 mutant (containing a point mutationresulting in a change of R-A at residue 15 of peptide 10 which isequivalent to residue 155 of the full length protein) which loses >60%of its inhibitory activity (see below), but retains its bindingfunction.

[0209] To determine if the inhibition of Cdk4 by p₂₁ ^(WAF1) could berelieved by the addition of a peptide 10 mutant, the R to A mutant(residue 15 of peptide 10) that was no longer an efficient inhibitor butstill displayed partial binding activity, increasing concentrations ofpeptide (1, 5, 17 & 34 μM) were added to cyclin D1-Cdk4 GST-Rbphosphorylation assay in the presence or a fixed concentration ofp21^(WAF1) (50 μM).

[0210] The experiment showed that increasing concentrations of mutantpeptide 10 were able to block the inhibitory activity of full lengthp21^(WAF1) suggesting that peptide 10 is binding at a site(s) whichblocks subsequent binding of p21^(WAF1) and is therefore functioningthrough a similar mechanism to the full length protein.

[0211] The Inhibitory Peptides are not Cyclin D1-Cdk4 Substrates

[0212] Unlike the p107 protein, which appears to inhibit Cdk4s abilityto phosphorylate pRb by acting as an alternative substrate (Zhu et al.,1995), p21^(WAF1) has not been reported to act as a substrate for thecyclin D1-Cdk4 complexes (and we confirm these observations FIG. 4).However, it is possible that by using p21^(WAF1) based peptides, insteadof full length protein, we have inadvertently generated phosphorylationsites which would not normally be exposed on the surface of the protein.Thus the peptides could be acting as competitive substrates as opposedto inhibitors of catalytic activity. Both peptide 2 and peptide 10contain a number of possible phosphorylation sites, and we have beenable to demonstrate that peptide 10 is a potential substrate for anumber of protein kinases (data not shown), including protein kinase C(PKC) which was used as a control kinase (FIG. 4). In fact, neitherpeptide 2 nor peptide 10 were substrates for cyclin D1-Cdk4 underconditions where 2.4 nMol of ³²P were incorporated per nMol of GST-Rb.However, under the same conditions peptide 10 was an extremely goodsubstrate for PKC with 0.82 nMol of ³²P being incorporated per nMol ofpeptide (FIG. 4). There was a low level of incorporation into peptide 2,but as this was also present in assays using lysate from uninfectedinsect cells it must be attributed to low levels of endogenous proteinkinase(s). Thus, it appears that the peptide inhibitors are notcompetitive substrates, but, are acting to block catalytic activity in amechanism similar to p21^(WAF1).

[0213] The Peptides are not Efficient Inhibitors of Cyclin B-Cdc2 KinaseActivity

[0214] Harper et al. (1995) have shown that p21^(WAF1) is not auniversal CDK inhibitor, but that it displays selectivity for the G1 andS-phase cyclin-CDK complexes. When they compared the ability ofp21^(WAF1) to inhibit Cyclin B-Cdc2, which acts at the G2/M transition,and cyclin D2-Cdk4, which functions during G1, they found that theI_(0.5) for inhibition of cyclin B-Cdc2 was >600-fold higher than theI_(0.5) for inhibition of cyclin D2-Cdk4 using purified recombinantproteins. We looked at the effect of adding our two cyclin D1-Cdk4inhibitory peptides to cyclin B-Cdc2 and Cdk2-cyclin E assays atconcentrations up to 20 μM and found that neither peptide 2 nor peptide10 had a significant effect on Cdc2-cyclin B histone H1 kinase activity.However, Cdk2-cyclin E was inhibited by peptide 10, showing that peptide10 can inhibit other G₁ cyclin-Cdk complexes. Thus, the p21^(WAF1) basedpeptide inhibitors appear to have equivalent specificity to the fulllength protein.

[0215] To determine if peptides 2 and 10 could inhibit cyclin B-Cdc2 andcyclin E-Cdk2 kinase activity assays were performed using Sf9 celllysates which were co-expressing human cyclin B and Cdc2. The conditionswere identical to those described in the Experimental Procedures forcyclin D1-Cdk4 except that histone H1 (0.5 μg/assay) was used as thesubstrate for cyclin B-Cdc2. Cyclin D1-Cdk4 cyclin B-Cdc2 and cyclinE-Cdk2 were assayed in the presence of increasing concentrations ofpeptide 2 (0.25, 3, 10 and 40 μM) and peptide 10 (0.1, 0.5, 5, 20 μM).

[0216] The Kinase Inhibitory Motif of Peptide 10 is Distinct from thePCNA Binding Site

[0217] We have shown that peptide 10 is an extremely potent inhibitor ofcyclin D1-Cdk4 activity, with an I_(0.5) of 0.1 mM which is 20-fold morepotent than peptide 2, a peptide derived from the region of p₂₁ ^(WAF1)previously associated with growth arrest (Chen et al., 1995; Nakanishiet al., 1995a). We have also shown that a peptide (peptide 4) whichspans the CDK interaction site of p21^(WAF1) (Goubin and Ducommun, 1995;Nakanishi et al., 1995a), although capable of binding to Cdk4 to form astable complex, has no detectable activity as a cyclin D1-Cdk4inhibitor. Peptide 10 therefore looks like the best candidate for thedevelopment of a small peptide mimetic with high efficacy. Peptide 10has previously been shown to form a specific high-affinity andreversible interaction with PCNA (Ball and Lane, 1996) and this peptideis sufficient to partially inhibit the function of PCNA during SV40replication giving 50% inhibition at a concentration of approximately 7mM (Warbrick et al., 1995). The PCNA interaction domain of p21^(WAF1)has been mapped and the important residues were found to be amino acids144-151 (QTSMTDFY; Warbrick et al., 1995; Ball and Lane, 1996). Althoughthe extreme C-terminal peptide (peptide 11) has amino acid residuesimportant for binding to and inhibiting Cdk4 (see FIGS. 1 and 2), itcannot bind PCNA (Warbrick et al., 1995; Ball and Lane, 1996). Theseresults indicate that the kinase inhibitory and PCNA binding motif inthe C-terminus of p21^(WAF1) are distinct, but it does not rule out thepossibility that an interaction between p21^(WAF1) and PCNA orcyclin/kinase may require some common amino acids. It is thereforeimportant to identify the precise inhibitory motif within the C-terminusof p21^(WAF1) and establish if it overlaps, or is distinct from, thePCNA interaction domain. To investigate this question we took twoapproaches; we synthesised, (i) a series of peptides that had beenshifted by 4 amino acids in either direction along peptide 10 (sizescan; FIG. 7), and (ii) a series of peptides based on peptide 10 whereeach residue was sequentially mutated to alanine (alanine scan; FIG. 6).The ability of the peptides, in each of these two series, to inhibitCdk4 activity in vitro was then determined. Using the size scan, wefound that the peptide inhibition activity required amino acids 156-160,while amino acids 148-155 were dispensable. This uncouples the kinaseinhibitory motif from the PCNA binding motif.

[0218] With the alanine scan we defined the critical residues forinhibition showing that a stretch of just S amino acids were essentialfor activity, with a single conservative point mutation at either of twohydrophobic residues completely abolishing peptide 10 inhibitoryactivity (FIG. 6). The essential amino acids are RRLIF (amino acids155-160) where the bold characters are essential for activity and theunderlined residue contributes significantly go inhibitory activity.

[0219] When tested in the peptide precipitation assay, mutation of thefirst R of this motif to A (aa 155 of full length p21^(WAF1)) partiallyretained its ability to bind both Cdk4 and cyclin D1 , whereas mutationsof L or F to A significantly decreased the affinity for Cdk4 and cyclinD1 , and mutations of the second R or the I had no effect on binding(data not shown). This is why the R-A mutant was used in competitionassays. The fact that a single point mutation in either of twohydrophobic residues (the L or F residues) completely abolishesinhibitory activity, suggested that inhibition was due to a specificinteraction at key hydrophobic residues. The mapping data also explainswhy both peptide 10 and peptide 11 are good inhibitors of cyclin D1-Cdk4activity (FIG. 2) as they both contain the inhibitory motif. Thus, itappears that the inhibitory portion of peptide 10 does not overlap withthe PCNA binding site as they have no amino acid residues in common.

[0220] A Single Amino Acid Substitution in Peptide 10 Makes it a MorePotent Inhibitor thus Approaching the Specific Activity of Full Lengthp21^(WAF1) Protein

[0221] Whilst carrying out the alanine scan experiments we noticed thatone of the mutant peptides (D-A at position 9 of peptide 10 or 149 ofthe full length protein; FIG. 6) appeared to make the peptide a betterinhibitor of cyclin D1-Cdk4 activity. We determined the I_(0.5) for thispeptide and compared it with peptide 10, full length purifiedhis-p21^(WAF1), and a peptide derived from the tumour suppressor proteinp16 INK4 which has recently been reported to inhibit cyclin D1-Cdk4activity in vitro and to prevent cell cycle progression (Fahraeus et al,1996). The D-A

[0222] mutation decreases the I_(0.5) from 100 nM to 46 nM (FIG. 7).Comparing this with the p16INK4-based peptide, which has an I_(0.5) of16.3 μM (FIG. 7), we have now produced a peptide which is approximately350-fold more active as a Cdk4 inhibitory compound. In fact, we nowbegin to approach the potency of p21^(WAF1) itself, which has an I_(0.5)of 11 nM in the insect cell lysate assay (FIG. 7). This value is in thesame range as the Ki of 40 nM for p21^(WAF1) obtained for the inhibitionof cyclin D1-Cdk4 in Sf9 cell lysates by Harper et al. (1995). Comparedto full length protein, the mutant peptide 10 has only a 3.5-fold lowerspecific activity as a kinase inhibitor in crude lysates. Why mutatingthe D-A in this position, which is well out side the domain shown to beessential for activity, reduces the I_(0.5) is not known. It seemslikely that it involves the presentation of the inhibitory motif, ratherthan a direct role for this residue in inhibition, as this mutation doesnot appear to increase the affinity of the peptide for either Cdk4 orcyclin D1 (data not shown).

[0223] The results indicate that peptide 10 could be used as a model onwhich to base small peptide mimetics of p21^(WAF1) and we have providedevidence that alterations in the peptides structure or presentation ofthe active residues may lead to the generation of a peptide inhibitorwhich approaches the potency of full length p21^(WAF1) as a cyclinD-Cdk4 inhibitor.

[0224] Results for the C-terminal peptide (Peptide 10)

[0225] An Eight Amino Acid Peptide is Sufficient to Inhibit CyclinD-Cdk4 Activity

[0226] Having identified residues which appeared to be critical for theinhibition of cyclin D1-Cdk4 by peptide 10, we determined if theseresidues were sufficient for inhibition, or if they had to be presentedwithin the context of a larger peptide. Strikingly, the eight amino acidpeptide, KRRLIFSK, retained the ability to completely inhibit cyclinD1-Cdk4 activity and prevent phosphorylation of pRb (FIG. 6). However,the 15 for the truncated peptide was approximately 1000-fold higher thanthat of the full length peptide (I_(0.5) for the truncated peptide wasapproximately 100 μM). This was not an unexpected result as otherstudies have shown loss of potency upon reducing the length ofbio-active peptides. However, it may be possible to improve the peptideinhibitory activity by manipulating the non-essential residues in amanner defined by Lin et al (1995) in an elegant series of experimentsaimed at minimising the atrial natriuretic peptide.

[0227] Peptide 10 Works in Cell Systems

[0228] The introduction of p21^(WAF1) cDNA into human brain, lung andcolon cancer cell lines leads to a suppression of cell growth (El-Deiryet al, 1993). In addition, during a radiation-induced G₁ arrest in humanfibroblasts p21^(WAF1) protein levels increase, in a p53-dependentmanner, leading to potent inhibition of the G₁ cyclin-CDKs and failureof the cells to enter S-phase (Dulic et al, 1994; Harper et al, 1995).In order for peptide 10 to function as a realistic template for thedesign of novel anti-proliferative drugs it must be able to mimicp21^(WAF1)'s CKI activity as a growth suppressor in a cellularbackground. We and others have recently shown that a 16 amino acidsequence from the homeodomain of the Antennapedia protein can act as acarrier for peptides with biological activity, translocating them acrossthe plasma membrane and allowing them to interact with their targetmolecules (F{dot over (a)}hraeus et al, 1996; Hall et al, 1996). Todetermine if peptide 10 retained its biological activity when introducedinto tissue culture cells, we synthesised it directly onto the carrierpeptide and added it to a culture of proliferating asynchronous humankerotinocyte-derived HaCaT cells. The linked peptide (designatedPeptide-I; FIG. 9) contained a mutation of M to A at position 7, thusabolishing its activity as a PCNA binding peptide (Warbrick et al, 1995;Ball and Lane, 1996), and allowed us to study PCNA-independent affectsof the peptide on normal cell cycle.

[0229] Peptide-I was added to the culture media at a concentration of 25μM, the cells were fixed 24 hours later, and then analysed byfluorescence-activated cell sorting (FACS). G₁, S- and G₂-phasedistribution of untreated and Peptide-I treated cells was assayed usingbromodeoxyuracil (BrdU). The number of cells entering S-phase in thepresence of Peptide-I was dramatically reduced and the G₁ populationshowed a concomitant increase. This suggests that Peptide-1 mimics theability of full length p21^(WAF1) to act as a growth suppressor byinducing a. G₁-cell cycle arrest.

[0230] In order to ascertain if Peptide-I was functioning as a growthinhibitor by preventing the phosphorylation of pRb in a manner analoguesto p21^(WAF1), we used serum starvation to produce a synchronouspopulation of HaCaT cells. Peptide-I was added to the cells at the sametime as they were released from serum starvation and samples fromtreated and untreated cells were taken over a 24 hour period. Thephosphorylation status of pRb was monitored by a gel mobility shiftassay. When serum was added to starved cells, pRb becamehyperphosphylated between 12 and 15 hours, but in the presence ofPeptide-I pRb remained hypophosphorylated. Thus, Peptide I causes aG₁-arrest in human HaCaT cells by preventing the phosphorylation of pRb.

[0231] We took an identical experimental approach to introduce, (i) thebio-active truncated peptide 10 and (ii) a control peptide 10 whichlacked essential residues for CDK inhibition, into HaCaT cells. We foundthat Peptide-II effectively promoted a G1-phase arrest and totallyprevented the phosphorylation of pRb when added at 50M (FIG. 9b).However Peptide-III, which lacked the last 4 amino acids of peptide 10(LIFS) had no detectable effect on the ability of HaCaT cells to enterS-phase. It is interesting that the truncated peptide 10 when coupled tocarrier peptide (Peptide-II) and introduced into cells is only 2-foldless active as a growth suppressor than Peptide-1 (see above for invitro data). Linking the truncated peptide 10 to the carrier peptide maypromote a more favourable inhibitory conformation, as the 10.5 forcarrier linked truncated peptide 10 (Peptide-II in vitro isapproximately 50-fold less than that of the free peptide 10 (data notshown).

[0232] Peptide 10 was added to Rb negative cells, and the resultssupport its mimicry of the full length protein, i.e. it can mimic itsbiolgical activity as a cell cycle inhibitor. Peptide 10 was found tocause cell cycle arrestin pRb negative, as well as pRb positive cells.Using Soas2 cells the introduction of peptide I (peptide 10 linked topenetratin and mutated to prevent PCNA binding) leads to an increase inthe population of cells in G1 phase.

[0233] Discussion

[0234] Synthetic peptides or peptido-mimetics are proving to be usefulin studying the biochemical regulation of enzymes and proteins, and alsoin providing models for the design of novel anti-proliferative agentstargeted to the enzymatic pathways amplified or proteins activated inhuman tumours (Powis, 1992; Gibbs and Oliff, 1994). Peptides which havebeen shown to effectively target components of the cell cycle machineryinclude: FTI, which inhibit farnesyl protein transferase preventing theactivation of Ras (Gibbs et al., 1994); Ras effector domain peptides,which can inhibit its biological function (Moodie and Wolfman, 1994;Rodriguez-Viciana et al., 1994); SH2/SH3 domain-harbouring polypeptides,which in theory should inhibit the growth of tumours with activatedtyrosine kinases (Pawson and Schlessinger, 1993; Yu et al., 1994), andp16INK4-derived peptides, which inhibit cyclin D-CDK complex activityand thereby activate pRb-dependent cell cycle arrest (F{dot over(a)}hraeus et al., 1996).

[0235] Inactivation of the tumour suppressor protein p53 is a commonevent in the development of human neoplasia (Hollstein et al., 1991).The p53 protein is a key player in an inducible cell cycle checkpointpathway activated in response to DNA-damage and nucleotide poolperturbation (Lane, 1992; Agarwal et al., 1995). Reactivation of thispathway could therefore provide a route to the discovery of novelanti-proliferative drugs. A variety of mechanisms could lead to thefunctional inactivation of the p53 pathway, including the inactivationof downstream effector molecules of p53, such as the cyclin-kinaseinhibitor p21^(WAF1) (Deng et al., 1995; Waldman et al., 1995). Recentdevelopments have shown that reactivation of the p53 pathway in somehuman tumours may be possible by activating the biochemical function ofthe endogenous mutant p53 protein (Halazonetis and Kandil, 1993; Hupp etal., 1993), possibly using small peptides as leads for drug design (Huppet al., 1995) or by reintroducing the wild type p53 gene usingadenovirus vectors (Eastham et al., 1995). However, in general, thepharmacological restoration of biochemical function to a protein thathas lost its normal-activity through mutation of its amino acid sequenceis more difficult than the inhibition of a biochemical function (Gibbsand Oliff, 1994). Thus, it may prove more productive to take alternativeapproaches to restore activity to the p53 pathway such as mimicking theinhibitory activity of the downstream effector molecule p21^(WAF1),which can by itself mediate growth arrest primarily through itsinteraction with the G1 cyclin-CDKs (El-Deiry et al., 1993; Eastham etal., 1995; Harper et al.; 1995).

[0236] Determining the minimal domain of p21^(WAF1) that can inhibit CDKfunction and whether such a domain can function in isolation with highefficiency are two important goals which must be achieved in order todetermine whether p21^(WAF1) will prove to be a realistic template foruse in anti-proliferative drug design research. Prior to our studies,the minimal sequence of p21^(WAF1) shown to inhibit CDK function invitro was the N-terminal domain (residues 1-75) (Luo et al., 1995)Whilst peptides derived from this N-terminal domain have recently beenshown to antagonise the ability of p21^(WAF1) to inhibit cyclin E-Cdk2complex activity suggesting that this domain interacts with the kinase(Chen et al., 1996), no data on the direct interaction of small peptideswith either cyclin or CDK has previously been presented. In addition, noevidence existed to suggest that a small peptide derived from p21^(WAF1)would in fact be biologically active as a CDK inhibitor. As the cyclinD1 -Cdk4 complexes and related isoforms are essential for progressionthrough G1-phase, we have used a series of small synthetic peptidesbased on the sequence of p21^(WAF1) to, (i) determine whether Cdk4inhibitory peptide-mimetics exist and if they are of high efficacy, and(ii) probe the mechanism by which the p21^(WAF1) protein inhibits cyclinD1-Cdk4 activity.

[0237] A Model for the Inhibition of Cyclin D1-Cdk4 by p21^(WAF1)

[0238] Two distinct peptides from the N-terminal domain of p21^(WAF1)interacted with either Cdk4 or cyclin D1 to form stable complexes. Onepeptide bound to Cdk4 but did not inhibit its activity, while the secondbound specifically to cyclin D1 and had potent inhibitory effects oncyclin D1-Cdk4 activity. The Cdk4 binding peptide 4 (residues 46-65)corresponded to a putative Cdk2 binding domain of p21^(WAF1) previouslydefined using p21^(WAF1) deletion constructs (Nakanishi et al., 1995a)and alanine mutation analysis (Goubin and Ducommun, 1995). We haveestablished that this region of p21^(WAF1) is, in fact, directlyinvolved in CDK binding, yet it has no Cdk4 inhibitory activity (FIGS. 2and 3). These data explain why certain N-terminally deleted p21^(WAF1)constructs, which still contain the CDK binding site, fail toefficiently inhibit cell growth (Nakanishi et al., 1995a).

[0239] The second N-terminal peptide, which bound to cyclin D1, potentlyinhibited cyclin D1-Cdk4 activity through a novel mechanism (see below).The mechanism of p21^(WAF1) inhibition of cyclin-CDK complexes is poorlyunderstood, as it has not been clear whether p21^(WAF1) protein inhibitsby cyclin and/or kinase subunit binding. Cdk2 binds very weakly top21^(WAF1) in the absence of cyclin, the affinity of the G1-CDKs forp21^(WAF1) being greatly increased if the CDK is associated with acyclin (Harper et al., 1995), suggesting that cyclins play an importantrole in p21^(WAF1) inhibition of CDK activity. However, whether a CKI,such as p21^(WAF1) and p27KIP1, can interact directly with cyclin is indispute (Toyoshima and Hunter, 1994; Harper et al., 1995). A recentstudy however, suggested that p21^(WAF1) can interact directly with anumber of cyclins in the absence of CDK (Fotedar et al., 1996). We showhere that a small peptide composed of residues 16-35 (peptide 2) forms astable complex with cyclin D1 and that this peptide alone is a potentinhibitor of Cdk4 activity, with an I_(0.5) of 2 mM. This peptide fallswithin the growth suppressor region (residues 17-71), described byNakanishi et al. (1995a) This is the first time that a putative cyclinbinding site on p21^(WAF1) has been identified and that a smallsynthetic peptide representing this domain has been shown to besufficient to mimic the full length p21^(WAF1) protein as a CDKinhibitor.

[0240] The fact that cyclin D1-Cdk4 activity can be inhibited byinteraction with the cyclin subunit alone, suggests either (i) thatconformational changes in cyclin D1 can lead to the inhibition of Cdk4catalytic activity, (ii) that peptide 2 interferes with the interactionof cyclin D1 with Cdk4 or (iii) that peptide 2 interferes with theinteraction of cyclin D1-Cdk4 with its substrate pRb.

[0241] Prospects for the design of small molecular mimetics ofp21^(WAF1) are more viable given that the cyclin D1-binding peptidealone can inhibit kinase function, indicating that the prior presence ofone p21^(WAF1) protein binding to the kinase subunit is not required forinhibition of kinase function. In addition, the amino acid residues thatare conserved between p21^(WAF1) and its close relative p27KIP1 (Polyaket al., 1994; Toyoshima and Hunter, 1994) are clustered within theN-terminal domain, with the regions corresponding to peptides 2 (65%identical) and peptide 4 (50 identical) containing the majority of theconserved amino acids. This suggests that inhibition of Cdk4 activity byinteraction with the cyclin D subunit may be a common mechanism employedby both p21^(WAF1) and p27KIP1.

[0242] A Novel p21^(WAF1) C-Terminal Cyclin D1-Cdk4 Inhibitory DomainDuring the course of our studies we also found that a peptide (peptide10) from the C-terminal domain of p21^(WAF1) was a potent inhibitor ofcyclin D1-Cdk4 activity in vitro. The inhibitory motif was identifiedand was distinct from the PCNA interacting site, which also resides inthe C-terminal domain of p21^(WAF1) (Chen et al., 1995; Luo et al.,1995; Warbrick et al., 1995; Ball and Lane, 1996). Our results are incontrast to previous studies which have found that cyclin-Cdk2inhibitory activity is confined solely to the N-terminal domain ofp21^(WAF1), when each half is expressed separately (Chen et al., 1995;Luo et al., 1995). The reasons for this discrepancy may include: (i) theuse of C-terminally his-tagged p21^(WAF1) in expression vectors forpurifying p21^(WAF1) constructs (Luo et al., 1995), which may haveaffected the local structure at the C-terminus of p21^(WAF1); (ii) thetransfection of constructs containing only the C-terminal half ofp21^(WAF1) (Chen et al., 1995; Luo et al., 1995) this may make foldinginto the correct native conformation difficult precluding identificationof the novel inhibitory domain; (iii) by using peptides, rather that theC-terminal constructs or full length p21^(WAF1) protein, we may haveexposed sites which would not be solvent exposed in native full lengthp21^(WAF1) protein; (iv) it is possible that there may be subtledifferences in the mechanism(s) used by p21^(WAF1) to inhibitcyclin-Cdk2 complexes and cyclin D1-Cdk4. Whether, the C-terminalinhibitory motif defines a novel physiologically relevant regulatorysite on p21^(WAF1) is currently being addressed. However, the potency ofpeptide 10 (I_(0.5)=0.1 mM, only 10-fold lower than full lengthp21^(WAF1) protein in these assays) and its ability to completelyinhibit cyclin D1-Cdk4, suggests to us that further studies on thisregion of full length p21^(WAF1) will be well worth pursuing.

[0243] Peptide 10 represents a potentially exciting lead for drug designas it is by far the most potent peptide inhibitor of CDK activitydiscovered to date, being >150-fold better than the recently identifiedpeptide mimetic of p16INK4 (Fahraeus et al., 1996) and 20-fold betterthan the N-terminal inhibitory p21^(WAF1)-derived-peptide which we havedescribed. The fact that the residues important for inhibitory activityare confined to a stretch of just five amino acids, suggests thatcontact at a single interface is sufficient to produce a highly potentinhibitor of the cyclin D1-Cdk4 activity, making this a realistictemplate for the design of small molecules which mimic p21^(WAF1)activity.

[0244] The fact that peptide 10 retains inhibitory activity when reducedto just eight amino acids (KRRLIFSK) improves its appeal as a templatefor rational drug design. In general protein-protein interfaces arerelatively large relying on the participation of between 10-30 contactside chains on each interface, with each region of contact often beingcomposed of residues which are dispersed throughout the primary aminoacid sequence (Davies et al, 1990; de Vos et al, 1992) However, there isevidence that in some cases only a small subset of these side chainsneed to be contacted for efficient binding to occur (Kelley andO'connel, 1993, Cunningham and Wells, 1994; Clackson and Wells, 1995).The discovery that a single eight amino acid peptide is alone sufficientto inhibit the activity of a critical G₁-cyclin-CDK preventing pRbphosphorylation and producing a G₁-cell cycle arrest in tissue culturecell systems, suggests that interaction at only a small subset ofcontact side chains is necessary for potent inhibition of cyclin D1-Cdk4activity at the G1-S phase boundary. This makes cyclin D1-Cdk4 arealistic and exciting target for the design of small syntheticcompounds which can at act as anti-proliferative agents.

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1 28 1 20 PRT Artificial Sequence Description of Artificial SequenceSynthesised 1 Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys GlySer Lys 1 5 10 15 Ala Cys Arg Arg 20 2 20 PRT Artificial SequenceDescription of Artificial Sequence Synthesised 2 Lys Ala Cys Arg Arg LeuPhe Gly Pro Val Asp Ser Glu Gln Leu Ser 1 5 10 15 Arg Asp Cys Asp 20 320 PRT Artificial Sequence Description of Artificial SequenceSynthesised 3 Ser Arg Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln GluAla Arg 1 5 10 15 Glu Arg Trp Asn 20 4 20 PRT Artificial SequenceDescription of Artificial Sequence Synthesised 4 Arg Glu Arg Trp Asn PheAsp Phe Val Thr Glu Thr Pro Leu Glu Gly 1 5 10 15 Asp Phe Ala Trp 20 520 PRT Artificial Sequence Description of Artificial SequenceSynthesised 5 Gly Asp Phe Ala Trp Glu Arg Val Arg Gly Leu Gly Leu ProLys Leu 1 5 10 15 Tyr Leu Pro Thr 20 6 20 PRT Artificial SequenceDescription of Artificial Sequence Synthesised 6 Leu Tyr Leu Pro Thr GlyPro Arg Arg Gly Arg Asp Glu Leu Gly Gly 1 5 10 15 Gly Arg Arg Pro 20 720 PRT Artificial Sequence Description of Artificial SequenceSynthesised 7 Gly Gly Arg Arg Pro Gly Thr Ser Pro Ala Leu Leu Gln GlyThr Ala 1 5 10 15 Glu Glu Asp His 20 8 20 PRT Artificial SequenceDescription of Artificial Sequence Synthesised 8 Ala Glu Glu Asp His ValAsp Leu Ser Leu Ser Cys Thr Leu Val Pro 1 5 10 15 Arg Ser Gly Glu 20 920 PRT Artificial Sequence Description of Artificial SequenceSynthesised 9 Pro Arg Ser Gly Glu Gln Ala Glu Gly Ser Pro Gly Gly ProGly Asp 1 5 10 15 Ser Gln Gly Arg 20 10 20 PRT Artificial SequenceDescription of Artificial Sequence Synthesised 10 Lys Arg Arg Gln ThrSer Met Thr Asp Phe Tyr His Ser Lys Arg Arg 1 5 10 15 Leu Ile Phe Ser 2011 20 PRT Artificial Sequence Description of Artificial SequenceSynthesised 11 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu IlePhe Ser 1 5 10 15 Lys Arg Lys Pro 20 12 5 PRT Artificial SequenceDescription of Artificial Sequence Motif 12 Arg Arg Leu Ile Phe 1 5 13 8PRT Artificial Sequence Description of Artificial Sequence Motif 13 LysArg Arg Leu Ile Phe Ser Lys 1 5 14 9 PRT Artificial Sequence SITE(2)..(3) Xaa may be any amino acid 14 Xaa Xaa Xaa Arg Arg Xaa Phe XaaXaa 1 5 15 16 PRT Artificial Sequence Description of Artificial SequenceCarrier peptide 15 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met LysTrp Lys Lys 1 5 10 15 16 20 PRT Artificial Sequence Description ofArtificial Sequence Synthesised 16 Pro Arg Ser Gly Glu Gln Ala Glu GlySer Pro Gly Gly Pro Gly Asp 1 5 10 15 Ser Gln Gly Arg 20 17 20 PRTArtificial Sequence Description of Artificial Sequence Synthesised 17Glu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg 1 5 1015 Lys Arg Arg Gln 20 18 20 PRT Artificial Sequence Description ofArtificial Sequence Synthesised 18 Gly Ser Pro Gly Gly Pro Gly Asp SerGln Gly Arg Lys Arg Arg Gln 1 5 10 15 Thr Ser Met Thr 20 19 20 PRTArtificial Sequence Description of Artificial Sequence Synthesised 19Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln Thr Ser Met Thr 1 5 1015 Asp Phe Tyr His 20 20 20 PRT Artificial Sequence Description ofArtificial Sequence Synthesised 20 Ser Gln Gly Arg Lys Arg Arg Gln ThrSer Met Thr Asp Phe Tyr His 1 5 10 15 Ser Lys Arg Arg 20 21 20 PRTArtificial Sequence Description of Artificial Sequence Synthesised 21Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 1015 Lys Arg Lys Pro 20 22 16 PRT Artificial Sequence Description ofArtificial Sequence Synthesised 22 Asp Phe Tyr His Ser Lys Arg Arg LeuIle Phe Ser Lys Arg Lys Pro 1 5 10 15 23 8 PRT Artificial SequenceDescription of Artificial Sequence Truncated peptide 23 Lys Arg Arg LeuIle Phe Ser Lys 1 5 24 36 PRT Artificial Sequence Description ofArtificial Sequence Synthesised 24 Lys Arg Arg Gln Thr Ser Ala Thr AspPhe Tyr His Ser Lys Arg Arg 1 5 10 15 Leu Ile Phe Ser Arg Gln Ile LysIle Trp Phe Gln Asn Arg Arg Met 20 25 30 Lys Trp Lys Lys 35 25 24 PRTArtificial Sequence Description of Artificial Sequence Synthesised 25Lys Arg Arg Leu Ile Phe Ser Lys Arg Gln Ile Lys Ile Trp Phe Gln 1 5 1015 Asn Arg Arg Met Lys Trp Lys Lys 20 26 30 PRT Artificial SequenceDescription of Artificial Sequence Synthesised 26 Arg Gln Thr Ser MetThr Asp Phe Tyr His Ser Lys Arg Arg Arg Gln 1 5 10 15 Ile Lys Ile TrpPhe Gln Asn Arg Arg Met Lys Trp Lys Lys 20 25 30 27 8 PRT ArtificialSequence Description of Artificial Sequence Synthesised 27 Gln Thr SerMet Thr Asp Phe Tyr 1 5 28 20 PRT Artificial Sequence Description ofArtificial Sequence Synthesised 28 Lys Arg Arg Gln Thr Ser Ala Thr AspPhe Tyr His Ser Lys Arg Arg 1 5 10 15 Leu Ile Phe Ser 20

1. A method of inhibiting the activity of a G1 cdk, the method includingcontacting said cdk with a substance that includes a peptide of 40 aminoacids or less, the peptide including the motif: Kxxrryfzp (wherein x maybe any amino acid, y and z may be hydrophobic, and each of theunderlined residues may be absent or different).
 2. A method accordingto claim 1 wherein y or/and z is, or are independently, any one of:alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan, methionine.
 3. A method according to claim 1, wherein saidpeptide is a fragment of p21 or an active portion or derivative thereof.4. A method according to claim 1, wherein said peptide consists ofresidues 16-35 of the p21^(WAF1) amino acid sequence or an activeportion or derivative thereof.
 5. A method according to claim 3 or claim4, wherein said peptide is a said active portion or a said derivativeand said active portion or said derivative has at least 80% identitywith p21 over a window of at least 5 amino acids.
 6. A method accordingto claim 1 wherein said peptide is coupled to a carrier molecule.
 7. Amethod according to claim 6, wherein the carrier molecule has thesequence RQIKIWFQNRRMKWKK.
 8. A method according to claim 1 wherein thepeptide binds to a G1 cyclin or a G1 cdk.
 9. An assay method for acompound with ability to modulate interaction or binding between apeptide as defined in claim 1 and a G1 cyclin and/or a G1 cdk, themethod including: (a) bringing into contact said peptide, a substanceincluding a said cyclin or an active portion or derivative thereof,and/or a substance including a said cdk or an active portion orderivative thereof, and a test compound, under conditions wherein, inthe absence of the test compound being an inhibitor of interaction orbinding of said peptide and one or more of said substances, said peptideand one or more of said substances interact or bind; and (b) determininginteraction or binding between said peptide and one or more of saidsubstances.
 10. A method according to claim 9, wherein a compound isadditionally tested for ability to modulate a p21-mediated effect onactivity of a G1 cdk.
 11. A method according to claim 1 or claim 10wherein the cdk activity includes Rb phosphorylation.
 12. A methodaccording to claim 1 or claim 10 wherein induction of cell cycle arrestis tested.